Methods for overcoming glucocorticoid resistance and for determining glucocorticoid resistance potential in cancer

ABSTRACT

The invention provides methods for overcoming glucocorticoid resistance of cancers by inhibition of CASP1. Also disclosed are diagnostic methods for determining glucocorticoid resistance potential by measuring expression level or promoter methylation status of CASP1 gene and/or NLRP3 gene.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. application Ser.No. 15/022,464, filed on Mar. 16, 2016, which is the U.S. National Phaseapplication under 35 U.S.C. § 371 of International Patent ApplicationNo. PCT/US2014/055824, filed on Sep. 16, 2014, and claims the benefit ofU.S. Provisional Patent Application No. 61/878,373, filed on Sep. 16,2013, all of which applications are incorporated herein by references intheir entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grants CA141762,CA021765, GM092666 and CA036401 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 1, 2019, isnamed 243734.000124_SL.txt and is 32,454 bytes in size.

FIELD OF THE INVENTION

The invention provides methods for overcoming glucocorticoid resistanceof cancers by inhibition of CASP1. Also disclosed are diagnostic methodsfor determining glucocorticoid resistance potential by measuringexpression level or promoter methylation status of CASP1 gene and/orNLRP3 gene.

BACKGROUND OF THE INVENTION

Glucocorticoids (GCs) are steroid hormones that regulate multiplephysiological processes involved in inflammation, immunity, metabolismand homeostatic functions. They exert their effects by binding to theglucocorticoid receptor (GR, NR3C1), triggering its activation andtranslocation to the nucleus, leading to transcriptional changesresponsible for diminished proliferative capacity and leukemia celldeath (Yudt and Cidlowski, 2002).

Synthetic glucocorticoids (e.g., Hydrocortisone (Plenadren, Cortef),Prednisone, Dexamethasone (Intensol), Methylprednisolone (Medrol),Prednisolone (Orapred, Pediapred, Prelone)) are widely prescribedmedications used to treat a variety of human diseases with aninflammatory component such as, e.g., asthma, allergies, chronicobstructive pulmonary disease (COPD), swelling, organ transplants,sarcoidosis, spinal cord injuries, ulcerative colitis, irritation,rheumatoid arthritis, chronic inflammatory demyelinating polyneuropathy,Addison's disease, multiple sclerosis (MS), and other autoimmunedisorders. However, success of these glucocorticoid treatments isfrequently hampered by resistance. For instance, glucocorticoidresistance in asthma has been estimated at 20% -50% (Barnes et al., Am.J. Respir. Critical Care Med., 1995, 152:5125-5142).

Glucocorticoids (e.g., prednisolone and dexamethasone) are also anessential component of curative therapy of acute lymphoblastic leukemia(ALL) and lymphomas. Advancements in the treatment of children with ALLhave led to five-year disease-free survival rates exceeding 85% (Pui etal., 2009). However, children whose ALL cells show in vitro resistanceto glucocorticoids have a significantly worse treatment outcome(disease-free survival) than patients whose ALL cells are sensitive toglucocorticoids (Den Boer et al., 2003; Dordelmann et al., 1999; Kasperset al., 1997; Pieters et al., 1991). Yet, little is known about themechanisms causing the leukemia cells from some patients to exhibit denovo resistance to glucocorticoids.

To find ways to overcome glucocorticoid resistance in acutelymphoblastic leukemia (ALL), lymphomas and other cancers is an urgentproblem.

Caspase-1 (CASP1, also known as Interleukin-lβ converting enzyme (ICE),apoptosis-related cysteine peptidase, IL-lβ convertase, P45 and IL1Bconvertase, and IL1BC), is an intracellular protease that is known tocleave the precursors of IL-lβ and IL-18 into active cytokines (Black etal., FEBS Lett, 247: 386-390 (1989); Kostura et al., Proc. Natl. Acad.Sci. U.S.A., 86:5227-5231 (1989)). Enzymatically active CASP1 is aheterotetramer composed of two subunits of p20 and two subunits of p10(20 kDa and 10 kDa molecular weight, respectively). These subunits arederived from a 45 kDa proenzyme (p45) by way of a p30 form, through anactivation mechanism that is autocatalytic (Thornberry et al., Nature,356, pp.768-774 (1992)). The CASP1 proenzyme has been divided intoseveral functional domains: a prodomain (pi 4), a p22/20 subunit, apolypeptide linker and a p10 subunit (Thornberry et al., Nature, 356,pp.768-774 (1992); Casano et al., Genomics, 20, pp. 474-481 (1994)).

CASP1 belongs to a family of cysteine proteases that cleave proteinsfollowing an aspartic acid residue. Produced as a pro-enzyme, CASP1requires removal of its CARD (caspase activation and recruitment domain)before it becomes an active enzyme (Schroder and Tschopp, 2010). CARDcleavage is mediated by the formation of large complexes termedinflammasomes, of which the most extensively characterized is theNLRP3-containing inflammasome. NLRP3 (encoded by NLRP3 gene) can beactivated by exposure to pathogen associated molecular pattern (PAMP) ordamage associated molecular pattern (DAMP) molecules, or by wholepathogens or environmental irritants (Schroder and Tschopp, 2010). Thereis also emerging evidence that the NLRP3 inflammasome can form inresponse to host-derived molecules, including extracellular ATP, glucoseor monosodium urate crystals (Mariathasan et al., 2006; Martinon et al.,2006; Schroder and Tschopp, 2010; Zhou et al., 2010). In vivo inductionof the NLRP3 inflammasome typically results in self-oligomerization,recruitment of the ASC (PYCARD) adaptor protein, and clustering andautoactivation of CASP1.

Human CASP1 has the following sequence (SEQ ID NO: 1, corresponds toGenBank Accession No. NP_150634.1), wherein residues 1-119 of correspondto the propeptide region and residues 120-404 correspond to the maturechain. The p20 and p10 subunits correspond to residues 120-297 (p20) andresidues 317-404 (p10), respectively.

(SEQ ID NO: 1) MADKVLKEKRKLFIRSMGEGTINGLLDELLQTRVLNKEEMEKVKRENATVMDKTRALIDSVIPKGAQACQICITYICEEDSYLAGTLGLSADQTSGNYLNMQDSQGVLSSFPAPQAVQDNPAMPTSSGSEGNVKLCSLEEAQRIWKQKSAEIYPIMDKSSRTRLALIICNEEFDSIPRRTGAEVDITGMTMLLQNLGYSVDVKKNLTASDMTTELEAFAHRPEHKTSDSTFLVFMSHGIREGICGKKHSEQVPDILQLNAIFNMLNTKNCPSLKDKPKVIIIQACRGDSPGVVWFKDSVGVSGNLSLPTTEEFEDDAIKKAHIEKDFIAFCSSTPDNVSWRHPTMGSVFIGRLIEHMQEYACSCDVEEIFRKVRFSFEQPDGRAQMPTTERVTLTRCFYL FPGH

See also the following GenBank Accession Nos.:

Alpha precursor isoforms: NP_150634.1 (as above); NP_001244047.1Beta precursor isoforms: NP_001214.1; NP_001244048.1Gamma precursor isoforms: NP_150635.1Delta precursor isoforms: NP_150636.1Epsilon precursor isoforms: NP_150637.1

Human NLRP3 has the following sequence (SEQ ID NO: 2; corresponds toGenBank Accession No. NP_004886.3), wherein residues 8-93 of correspondto the pyrin death domain found in NALP proteins and residues 220-389correspond to the NACHT domain and resides 575-891 correspond toLeucine-rich repeats (LRRs), ribonuclease inhibitor domain respectively.

(SEQ ID NO: 2) MKMASTRCKLARYLEDLEDVDLKKFKMHLEDYPPQKGCIPLPRGQTEKADHVDLATLMIDFNGEEKAWAMAVWIFAAINRRDLYEKAKRDEPKWGSDNARVSNPTVICQEDSIEEEWMGLLEYLSRISICKMKKDYRKKYRKYVRSRFQCIEDRNARLGESVSLNKRYTRLRLIKEHRSQQEREQELLAIGKTKTCESPVSPIKMELLFDPDDEHSEPVHTVVFQGAAGIGKTILARKMMLDWASGTLYQDRFDYLFYIHCREVSLVTQRSLGDLIMSCCPDPNPPIHKIVRKPSRILFLMDGFDELQGAFDEHIGPLCTDWQKAERGDILLSSLIRKKLLPEASLLITTRPVALEKLQHLLDHPRHVEILGFSEAKRKEYFFKYFSDEAQARAAFSLIQENEVLFTMCFIPLVCWIVCTGLKQQMESGKSLAQTSKTTTAVYVFFLSSLLQPRGGSQEHGLCAHLWGLCSLAADGIWNQKILFEESDLRNHGLQKADVSAFLRMNLFQKEVDCEKFYSFIHMTFQEFFAAMYYLLEEEKEGRTNVPGSRLKLPSRDVTVLLENYGKFEKGYLIFVVRFLFGLVNQERTSYLEKKLSCKISQQIRLELLKWIEVKAKAKKLQIQPSQLELFYCLYEMQEEDFVQRAMDYFPKIEINLSTRMDHMVSSFCIENCHRVESLSLGFLHNMPKEEEEEEKEGRHLDMVQCVLPSSSHAACSHGLVNSHLTSSFCRGLFSVLSTSQSLTELDLSDNSLGDPGMRVLCETLQHPGCNIRRLWLGRCGLSHECCFDISLVLSSNQKLVELDLSDNALGDFGIRLLCVGLKHLLCNLKKLWLVSCCLTSACCQDLASVLSTSHSLTRLYVGENALGDSGVAILCEKAKNPQCNLQKLGLVNSGLTSVCCSALSSVLSTNQNLTHLYLRGNTLGDKGIKLLCEGLLHPDCKLQVLELDNCNLTSHCCWDLSTLLTSSQSLRKLSLGNNDLGDLGVMMFCEVLKQQSCLLQNLGLSEMYFNYETKSALETLQEEKPELTVVFEPSW

See also the following GenBank Accession Nos.:

Isoform a: NP_004886.3 (as above); NP_001073289.1

Isoform b: NP_899632.1 Isoform c: NP_001120933.1 Isoform d:NP_001120934.1 Isoform e: NP_001230062.1 SUMMARY OF THE INVENTION

As specified in the Background Section, there is a great need in the artto find ways to overcome glucocorticoid resistance in acutelymphoblastic leukemia (ALL), lymphomas and other diseases. The presentinvention addresses this and other needs by providing methods forovercoming glucocorticoid resistance based on inhibition of CASP1.

Specifically, in one embodiment, the invention provides a method ofsensitizing a cancer cell to glucocorticoid-induced apoptosis or celldeath, wherein said cell is resistant to glucocorticoid-inducedapoptosis or cell death, comprising contacting the cell with aneffective amount of an inhibitor of CASP1.

In another embodiment, the invention provides a method of killing acancer cell, wherein said cell is resistant to glucocorticoid-inducedapoptosis or cell death, comprising contacting the cell with an amountof an inhibitor of CASP1 that is effective to sensitize the cell toglucocorticoid-induced apoptosis or cell death and further comprisingcontacting the cell with an effective amount of a glucocorticoid. In onespecific embodiment, the method further comprises contacting the cellwith a second agent for inducing apoptosis or cell death.

In one embodiment of the above methods, the cancer cell is in a patient.

In one embodiment of the above methods, the inhibitor of CASP1 directlyinhibits expression or function of CASP1. In another embodiment of theabove methods, the inhibitor of CASP1 directly inhibits expression orfunction of NLRP3. In yet another embodiment of the above methods, theinhibitor of CASP1 inhibits NLRP3 inflammasome formation or NLRP3inflammasome activity. In one specific embodiment of the above methods,the inhibitor of CASP1 is selected from the group consisting ofz-VAD-DCB, Ac-YVAD-CHO (SEQ ID NO: 18), Ac-YVAD-chloromethylketone (SEQID NO: 19), cytokine response modifier A (crmA), Pralnacasan (VX-740),IDN-6556, VX-765, VRT-043198, ML132, and SNAP.

In one embodiment of the above methods, the glucocorticoid is selectedfrom the group consisting of prednisolone, dexamethasone,hydrocortisone, prednisone, methylprednisolone, cortisol, budesonide,and combinations, enantiomers, optical isomers, diastereomers, N-oxides,crystalline forms, hydrates, metabolites or pharmaceutically acceptablesalts thereof.

In a separate embodiment, the invention provides a method of treating aglucocorticoid-resistant cancer in a subject (e.g., human) in needthereof which method comprises administering to the subject (i) atherapeutically effective amount of an inhibitor of CASP1 and (ii) atherapeutically effective amount of a glucocorticoid. In one embodiment,the therapeutically effective amount of the inhibitor of CASP1 is anamount that is effective to sensitize cancer cells within the subject toglucocorticoid-induced apoptosis or cell death. In one embodiment, thecancer is selected from the group consisting of acute lymphoblasticleukemia (ALL), acute myeloblastic leukemia (AML), lymphoma,osteosarcoma, small-cell lung cancer, breast cancer, brain cancer, andmultiple myeloma. In one embodiment, the method further comprisesadministering an additional anti-cancer treatment to the subject (e.g.,a chemotherapy or a radiation therapy).

In another embodiment, the invention provides a method of treating aglucocorticoid-resistant asthma in a subject (e.g., human) in needthereof which method comprises administering to the subject (i) atherapeutically effective amount of an inhibitor of CASP1 and (ii) atherapeutically effective amount of a glucocorticoid.

In one embodiment of the above treatment methods, the inhibitor of CASP1and the glucocorticoid are administered simultaneously. In oneembodiment of the above treatment methods, the inhibitor of CASP1 andthe glucocorticoid are administered in the same composition. In oneembodiment of the above treatment methods, the inhibitor of CASP1 andthe glucocorticoid are administered sequentially.

In one embodiment of the above treatment methods, the inhibitor of CASP1directly inhibits expression or function of CASP1. In another embodimentof the above treatment methods, the inhibitor of CASP1 directly inhibitsexpression or function of NLRP3. In yet another embodiment of the abovetreatment methods, the inhibitor of CASP1 inhibits NLRP3 inflammasomeformation or NLRP3 inflammasome activity. In one specific embodiment ofthe above treatment methods, the inhibitor of CASP1 is selected from thegroup consisting of z-VAD-DCB, Ac-YVAD-CHO (SEQ ID NO: 18),Ac-YVAD-chloromethylketone (SEQ ID NO: 19), cytokine response modifier A(crmA), Pralnacasan (VX-740), IDN-6556, VX-765, VRT-043198, ML132, andSNAP.

In one embodiment of the above treatment methods, the glucocorticoid isselected from the group consisting of prednisolone, dexamethasone,hydrocortisone, prednisone, methylprednisolone, cortisol, budesonide,and combinations, enantiomers, optical isomers, diastereomers, N-oxides,crystalline forms, hydrates, metabolites or pharmaceutically acceptablesalts thereof.

In a separate embodiment, the invention provides a pharmaceuticalcomposition comprising a CASP1 inhibitor, a glucocorticoid, and apharmaceutically acceptable carrier or excipient. In one embodiment, theCASP1 inhibitor is selected from the group consisting of z-VAD-DCB,Ac-YVAD-CHO (SEQ ID NO: 18), Ac-YVAD-chloromethylketone (SEQ ID NO: 19),cytokine response modifier A (crmA), Pralnacasan (VX-740), IDN-6556,VX-765, VRT-043198, ML132, and SNAP. In one embodiment, theglucocorticoid is selected from the group consisting of prednisolone,dexamethasone, hydrocortisone, prednisone, methylprednisolone, cortisol,budesonide, and combinations, enantiomers, optical isomers,diastereomers, N-oxides, crystalline forms, hydrates, metabolites orpharmaceutically acceptable salts thereof.

In a separate embodiment, the invention provides a method fordetermining whether a cancer in a subject (e.g., human) is likely to beresistant to a glucocorticoid treatment, said method comprising thesteps of:

(a) determining the expression level of CASP1 gene and/or NLRP3 gene incancer cells from the subject;(b) comparing the expression level of each gene determined in step (a)with a corresponding control expression level for that gene, and(c) (i) identifying that the cancer in the subject is likely to beresistant to the glucocorticoid treatment when the expression level ofCASP1 gene and/or NLRP3 gene in step (a) is increased by at least 1.5fold as compared to the corresponding control expression level or (ii)identifying that the cancer in the subject is not likely to be resistantto the glucocorticoid treatment when the expression level of CASP1 geneand/or NLRP3 gene in step (a) is not increased or is increased by lessthan 1.5 fold as compared to the corresponding control expression level.

In one embodiment, the corresponding control expression level is theexpression level of the same gene in similarly processed cancer cells ofthe same type which are sensitive to glucocorticoid treatment. Inanother embodiment, the corresponding control expression level is apredetermined standard. In one embodiment, the expression level of CASP1gene and/or NLRP3 gene is determined using a method selected from thegroup consisting of amplification-based assays, hybridization-basedassays, flap-endonuclease-based assays, and direct mRNA capture.

In a separate embodiment, the invention provides a method fordetermining whether a cancer in a subject is likely to be resistant to aglucocorticoid treatment, said method comprising the steps of:

(a) determining the methylation level of CASP1 gene promoter and/orNLRP3 gene promoter in cancer cells from the subject;(b) comparing the methylation level of each gene promoter determined instep (a) with a corresponding control methylation level for that genepromoter, and(c) (i) identifying that the cancer in the subject is likely to beresistant to glucocorticoid treatment when the methylation level ofCASP1 gene promoter and/or NLRP3 gene promoter in step (a) is decreasedby at least 1.5 fold as compared to the corresponding controlmethylation level or (ii) identifying that the cancer in the subject isnot likely to be resistant to glucocorticoid treatment when themethylation level of CASP1 gene promoter and/or NLRP3 gene promoter instep (a) is not increased or is increased by less than 1.5 fold ascompared to the corresponding control methylation level.

In one specific embodiment, the CASP1 gene promoter comprises thesequence GTCGGGGAAGGTTTTGAGAAAGAAGGGTCCCTGGACAAGAACCTTGTCATTTTCTGAGTGGCCGGTACCGAAAAGAGAGGAGGGAAGAACACACTGACTTTGACTTTCATACG AAGCGGAAG (SEQID NO: 3). In one specific embodiment, the NLRP3 gene promoter comprisesthe sequence CTCCTTTGACTTCAACTCCTTATCACTTCTCAAACAGGTTACAGTATCGGGGCATTAGTTGCCCTGTTTTTAAAAGAACGACTACCCAGTTCTACCGTAGCACTTCACCAACAAG TGGCATT (SEQID NO: 4). In one embodiment, the corresponding control methylationlevel is the methylation level of the same gene promoter in similarlyprocessed cancer cells of the same type which are sensitive toglucocorticoid treatment. In another embodiment, the correspondingcontrol methylation level is a predetermined standard. In oneembodiment, the methylation level is determined using a method selectedfrom the group consisting of methods based on the use ofmethylation-sensitive restriction endonucleases, bisulfite DNAsequencing, hybridization following bisulfite conversion, RestrictionLandmark Genome Scanning (RLGS), and Methylation-SensitiveRepresentational Difference Analysis (MS-RDA).

In one embodiment of the above diagnostic methods, the method comprisesobtaining the cancer cells from the subject prior to step (a). In oneembodiment, the cancer cells are derived from peripheral blood. Inanother embodiment, the cancer cells are derived from bone marrow. Inone embodiment, the cancer is selected from the group consisting ofacute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML),lymphoma, osteosarcoma, small-cell lung cancer, breast cancer, braincancer, and multiple myeloma.

In one embodiment of the above diagnostic methods, the subjectidentified as having cancer which is likely to be resistant toglucocorticoid treatment is treated with a combination of an inhibitorof CASP1 and a glucocorticoid. In one embodiment, the inhibitor of CASP1directly inhibits expression or function of CASP1. In anotherembodiment, the inhibitor of CASP1 directly inhibits expression orfunction of NLRP3. In yet another embodiment, the inhibitor of CASP1inhibits NLRP3 inflammasome formation or NLRP3 inflammasome activity. Inone specific embodiment, the inhibitor of CASP1 is selected from thegroup consisting of z-VAD-DCB, Ac-YVAD-CHO (SEQ ID NO: 18),Ac-YVAD-chloromethylketone (SEQ ID NO: 19), cytokine response modifier A(crmA), Pralnacasan (VX-740), IDN-6556, VX-765, VRT-043198, ML132, andSNAP.

In one embodiment of the above diagnostic methods, the subjectidentified as having cancer which is likely to be resistant toglucocorticoid treatment is treated with increased doses of aglucocorticoid (e.g., 1.5 fold increased doses of a glucocorticoid).

In another embodiment of the above diagnostic methods, the subjectidentified as having cancer which is likely to be resistant toglucocorticoid treatment is treated with a non-glucocorticoidchemotherapeutic and/or radiation.

In one embodiment of the above diagnostic methods, the subjectidentified as having cancer which is not likely to be resistant toglucocorticoid treatment is treated with a glucocorticoid.

In one embodiment of the above diagnostic methods, the glucocorticoidused for treatment is selected from the group consisting ofprednisolone, dexamethasone, hydrocortisone, prednisone,methylprednisolone, cortisol, budesonide, and combinations, enantiomers,optical isomers, diastereomers, N-oxides, crystalline forms, hydrates,metabolites or pharmaceutically acceptable salts thereof.

In a separate embodiment, the invention provides a kit for diagnosinglikelihood of cancer resistance to glucocorticoid treatment, said kitcomprising one or more pairs of oligonucleotides directed toward CASP1and/or NLRP3 nucleic acid, wherein said pairs of oligonucleotides can beused to determine the expression level of CASP1 gene and/or NLRP3 gene.In one embodiment, the kit further comprises a detection means. In oneembodiment, the kit further comprises an amplification means. In oneembodiment, the kit further comprises one or more pairs of controloligonucleotides. In one embodiment, the kit further comprisesinstructions for use.

In another embodiment, the invention provides a kit for diagnosinglikelihood of cancer resistance to glucocorticoid treatment, said kitcomprising antibodies directed toward CASP1 protein and/or NLRP3protein, wherein said antibodies can be used to determine the expressionlevel of CASP1 and/or NLRP3 protein. In one embodiment, the kit furthercomprises a detection means. In one embodiment, the kit furthercomprises one or more control antibodies. In one embodiment, the kitfurther comprises instructions for use.

In another embodiment, the invention provides a kit for diagnosinglikelihood of cancer resistance to glucocorticoid treatment, said kitcomprising means for detecting promoter methylation of CASP1 gene and/orNLRP3 gene. In one embodiment, the kit further comprises instructionsfor use.

These and other aspects of the present invention will be apparent tothose of ordinary skill in the art in the following description, claimsand drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent application file contains at least one drawing executed incolor. Copies of this patent application with color drawing(s) will beprovided by the Office upon request and payment of the necessary fee.

FIGS. 1A-I demonstrate that glucocorticoid resistant leukemia cells havehigher expression of CASP1 and NLRP3. Primary leukemia cells wereobtained by bone marrow aspirates from 444 patients with newly diagnosedacute lymphoblastic leukemia, then isolated and analyzed for theirsensitivity to prednisolone using the MTT assay (see Example 1, Methods)(Holleman et al., 2004). Distributions of measured LC50 values are shownfor the three independent cohorts of patients; sensitive and resistantleukemias are highlighted in green and red, respectively (panels A-C).CASP1 (panels D-F) and NLRP3 (panels G-I) expression was significantlyhigher in glucocorticoid resistant leukemia cells from these threecohorts of newly diagnosed patients. Exact Wilcoxon Mann-Whitney RankSum test p-values are shown for panels D-I, with Stouffer's Z-scoremethod meta-analysis p-values shown above panels D-F and panels G-I asdescribed in methods section.

FIGS. 2A-G demonstrate that hypo-methylation of the CASP1 and NLRP3promoter region is associated with higher CASP1 and NLRP3 expression inleukemia cells. In both patient cohorts for whom DNA was available forDNA methylation analysis (St. Jude Protocols XV and XVI), lower levelsof CASP1 (panels A, B and C) and NLRP3 (panels D, E and F) methylationwere found in leukemia cells with higher expression of CASP1 and NLRP3.For both CASP1 and NLRP3 methylation status, the DNA methylation site(CpG) was within 100 basepairs of the transcription start site (FIG. 6).k-means clustering analysis (▴ represents k-means identified group A, ●represents k-means identified group B, pink and blue squares representk-means identified centers for group A and B respectively) utilizingonly CASP1 and NLRP3 methylation status significantly discriminatedsensitive leukemias (green symbols, higher methylation) from resistantleukemias (red symbols, lower methylation) in both St. Jude Protocol XVand St. Jude Protocol XVI (panel G and FIG. 5) patients. Exact WilcoxonMann-Whitney Rank Sum test p-values are shown for panels A, B, D, E,with Stouffer's Z-score method meta-analysis p-values shown above panelsA-B and panels D-E.

FIGS. 3A-E show that CASP1 cleaves the glucocorticoid receptor andincreases resistance to glucocorticoids with an overarching schematicpresented in panel D. Bioinformatic analysis of the glucocorticoidreceptor (panel A, top) revealed a putative CASP1 cleavage site (LLID)(SEQ ID NO: 20) in the glucocorticoid receptor (NR3C1) transactivationdomain that is similar to a previously reported CASP1 cleavage site inthe androgen receptor, a close structural and functional homolog to theglucocorticoid receptor. Enzymatic assays revealed that recombinantCASP1 cleaves the GR and that this cleavage was inhibited by a CASP1tetrapeptide inhibitor (panel A, bottom). Site directed mutagenesis ofNR3C1 at the putative cleavage site (LLID motif) (SEQ ID NO: 20) blockedCASP1 cleavage at this location and revealed a secondary CASP1 cleavagesite more proximal to the carboxy terminus of the protein, as evidencedby a smaller enzymatic product (panel B). Further inhibition studiesshowed that small molecule inhibitors VX-765 and VRT-043198 (panel C)can also inhibit CASP1 cleavage of NR3C1, with VRT-043198 having higherinhibitory activity. Enforced expression of CASP1 in a human leukemiacell line increased resistance to prednisolone and dexamethasone afteractivation of the NLRP3 inflammasome (by the addition of LPS and ATP).Nalm6 cells were transduced with a lentivirus containing full lengthCASP1 and puromycin N-acetyl-transferase or puromycinN-acetyl-transferase alone (Control). Cells were puromycin selected andthen their sensitivity to prednisolone and dexamethasone (panel E) wasmeasured using the MTT assay, in the presence(+) and absence(−) ofinflammasome activation (LPS/ATP). The inset in panel E is a westernblot depicting the lower GR levels in cells over expressing CASP1 (24hours after activation of CASP1). FIG. 3A discloses SEQ ID NOS 23-24,respectively, in order of appearance. FIG. 3B discloses “LLID” and“AAAA” as SEQ ID NOS 20 and 25, respectively.

FIGS. 4A-B show that transcriptional modulation induced byglucocorticoids is blunted by CASP1. Cells expressing CASP1 or the emptyvector (control cells) were treated with or without prednisolone todetermine if CASP1 expression and activation alters transcriptionalactivation or repression of glucocorticoid response genes. Genome-widegene expression was measured (panels A and B) and fold-differences inthe expression of individual genes (with or without prednisolonetreatment) were compared between control cells treated with prednisolonecompared to the same cells not treated with prednisolone or in cellswith enforced expression and activation of CASP1 treated withprednisolone or not. The top 25 genes activated (blue bars) and the top25 repressed (green bars) by prednisolone in untreated control cells areshown, with the fold-change in CASP1 overexpressing cells also shown bythe adjacent gold bars. Additionally, control and CASP1 overexpressingcells were treated with and without prednisolone and LPS/ATP and proteinlysates were collected for determination of Bim protein levels, a knownglucocorticoid response gene (panel A inset).

FIG. 5 shows that k-means clustering analysis (▴ represents k-meansidentified group A, ● represents k-means identified group B, pink andblue squares represent k-means identified centers for group A and Brespectively) utilizing only CASP1 and NLRP3 methylation statussignificantly discriminated sensitive leukemias (green symbols, highermethylation) from resistant leukemias (red symbols, lower methylation)in both St. Jude Protocol XV and St. Jude Protocol XVI patients.

FIG. 6 shows DNA methylation analysis probe locations for CASP1 andNLRP3 relative to the transcription start sites of these genes. Thespecific base analyzed is shown in square brackets with the genomiccontext surrounding this site. FIG. 6 discloses SEQ ID NOS 26-27,respectively, in order of appearance.

FIG. 7 is a schematic diagram depicting overarching pathway andphenotypic consequences in patients.

FIG. 8 shows that CASP1 and NLRP3 expression is higher in leukemia cellsat the time of disease relapse. Expression levels of CASP1 (panel A) andNLRP3 (panel B) in leukemia cells were obtained from 49 patients atdiagnosis and at the time of relapse. Quantile normalized measures ofgene expression revealed significantly higher expression of NLRP3 andCASP1 at relapse. Paired t-test values are shown for panels A and B.

FIGS. 9A-D show that knockdown or inhibition of CASP1 reverses GRcleavage and prednisolone resistance. NALM-6 cells overexpressing CASP1were stably transduced with lentiviral vectors (Sigma-Aldrich, St.Louis, Mo.) containing non-targeting scrambled hairpin (shNT, Sequence:CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTT (SEQ ID NO:13)) or shRNA hairpins targeting CASP1 (shCASP1, Sequence:CCGGCTACAACTCAATGCAATCTTTCTCGAGAAAGATTGCATTGAGTTGTAGTTTTT (SEQ ID NO:14)). The two cell lines were cultured for 48 hours in the presence andabsence of LPS/ATP to allow for the activation of CASP1, andsubsequently analyzed on Western blot for the expression levels of CASP1and glucocorticoid receptor (GR) (Panel A). The quantified signalintensities of 90-kDa GR and 20-kDa active CASP1 normalized for theloading control HSP90, are shown in the bar graph (Panel A). Afteractivation of CASP1 (Panel A, lane 2 and 4), the level of the GR isapproximately 2-fold lower in scrambled hairpin cells (Panel A, lane 4),whereas cells in which CASP1 had been knocked down via the shCASP1showed minimal degradation of the GR (Panel A, lane 2). The sensitivity(LC50) of shCASP1 and shNT cells to prednisolone was determined by MTTassays (Panel B). In the absence of CASP1 activation (−LPS/ATP), thePRED-LC50 of shCASP1 and shNT cells were comparable (Panel B, brown andblue bars). However, upon activation of CASP1 (+LPS/ATP), shCASP1 cellswere 44-fold more sensitive to prednisolone (lower LC50) compared toshNT cells (Panel B, light brown and teal bars). Error bars representS.E.M; n=4 replicate experiments. Transduction of CrmA (catalyticinhibitor of CASP1 protein; nucleotide sequence shown in SEQ ID NO: 15)or GFP in CASP1 overexpressing cells showed effects similar to knockdownof CASP1 by shRNA. In the absence of LPS/ATP33 induced CASP1 activation,the PRED-LC50 for CrmA-expressing cells and GFP-expressing cells werecomparable (Panel D, brown and blue bars). However, upon activation ofCASP1 by LPS/ATP, CrmA blocked CASP1 induced GR cleavage (Panel C) andincreased GC sensitivity by 43-fold compared to the GFP expressingcontrol cells (Panel D, light brown and teal bars).

FIG. 10 shows that expression of CASP1 cleavage site double mutagenizedGR reverses CASP1-induced prednisolone resistance. NALM-6 cellsoverexpressing CASP1 co-transfected with either non-targeting scrambledhairpin (shNT; SEQ ID NO: 13) or shRNA hairpins targeting CASP1(shCASP1; SEQ ID NO: 14) were stably transduced with lentiviral vectors(Addgene plasmid 25890; Sigma-Aldrich, St. Louis, Mo.) containing awild-type glucocorticoid receptor (SEQ ID NO: 16) or a glucocorticoidreceptor in which both CASP1 cleavage sites had been eliminated (doublemutagenized GR, DM-GR (SEQ ID NO: 17)). The sensitivity (LC50) of thesecells to prednisolone was determined by MTT assays, revealing that cellsoverexpressing a GR without CASP1 cleavage sites remained sensitive toglucocorticoids when CASP1 was over-expressed. The inset is a westernblot depicting recombinant GR protein levels (wild-type, or doublemutagenized), in cells at time of MTT drug sensitivity assays.

FIG. 11 depicts GenBank Accession Nos.: Alpha precursor isoforms:NP_150634.1; NP_001244047.1; Beta precursor isoforms: NP_001214.1,NP_001244048.1; Gamma precursor isoforms: NP_150635.1; Delta precursorisoforms: NP_150636.1; and Epsilon precursor isoforms: NP_150637.1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on an unexpected observation by theinventors that primary acute lymphoblastic leukemia (ALL) cells thatexhibited de novo resistance to glucocorticoids had higher expression ofCASP1 and its activator NLRP3 (NLR family, pyrin domain containing 3).The present inventors also found significantly lower promotermethylation of these two genes in leukemia cells exhibiting higherexpression of CASP1 and NLRP3. As demonstrated in the Examples section,below, the mechanism involves CASP1 cleavage of the glucocorticoidreceptor (GR) in its transactivation domain, thereby reducing cellularlevels of functional GR and altering cellular response to glucocorticoidtreatment.

Definitions

The term “glucocorticoid resistant cancer” is used herein to refer to acancer in which the cancer cells are resistant to being killed by theconcentrations of a glucocorticoid that are used to kill cells in adrug-sensitive cancer of the same type. Non-limiting examples ofglucocorticoids for which resistance can occur in cancer cells includeprednisolone, dexamethasone, hydrocortisone, prednisone,methylprednisolone, cortisol, budesonide, and combinations, enantiomers,optical isomers, diastereomers, N-oxides, crystalline forms, hydrates,metabolites or pharmaceutically acceptable salts thereof. Non-limitingexamples of cancers in which glucocorticoid resistance is frequentlyobserved include acute lymphoblastic leukemia (ALL, including both B-and T-lineage ALL), acute myeloblastic leukemia (AML), lymphoma,osteosarcoma, small-cell lung cancer, breast cancer, brain cancer, andmultiple myeloma.

The relative resistance of a cancer cell to a glucocorticoid may bedetermined by calculating the drug concentration that is lethal to 50%of the cells (LC50). See, e.g., Holleman et al., 2004. As disclosed inthe Example 1, below, with respect to resistance of ALL to prednisolone,glucocorticoid resistant ALL was defined as having an LC50 greater thanor equal to 64 μM, whereas glucocorticoid sensitive cases were definedas having an LC50 less than 0.1 μM (for LC50 measured using the methodof Holleman et al., 2004).

As used herein, the terms “CASP1 inhibitor” and “inhibitor of CASP1”encompass direct inhibitors of expression and/or function of CASP1 aswell as direct inhibitors of expression and/or function of NLRP3,inhibitors of NLRP3 inflammasome formation and inhibitors of NLRP3inflammasome activity.

The term “a control level” as used herein encompasses predeterminedstandards (e.g., a published value in a reference) as well as levelsdetermined experimentally in similarly processed samples from controlcancer cells of the same type which are sensitive to glucocorticoidtreatment (preferably, sensitive to treatment with the sameglucocorticoid predisposition to resistance to which is being tested).As used herein, the term “similarly processed” refers to samples whichhave been obtained using the same protocol.

The term “about” means within an acceptable error range for theparticular value as determined by one of ordinary skill in the art,which will depend in part on how the value is measured or determined,i.e., the limitations of the measurement system. For example, “about”can mean within an acceptable standard deviation, per the practice inthe art. Alternatively, “about” can mean a range of up to ±20%,preferably up to ±10%, more preferably up to ±5%, and more preferablystill up to ±1% of a given value. Alternatively, particularly withrespect to biological systems or processes, the term can mean within anorder of magnitude, preferably within 2-fold, of a value. Whereparticular values are described in the application and claims, unlessotherwise stated, the term “about” is implicit and in this context meanswithin an acceptable error range for the particular value.

In the context of the present invention insofar as it relates to any ofthe disease conditions recited herein, the terms “treat”, “treatment”,and the like mean to relieve or alleviate at least one symptomassociated with such condition, or to slow or reverse the progression ofsuch condition. Within the meaning of the present invention, the term“treat” also denotes to arrest, delay the onset (i.e., the period priorto clinical manifestation of a disease) and/or reduce the risk ofdeveloping or worsening a disease. E.g., in connection with cancer theterm “treat” may mean eliminate or reduce a patient's tumor burden, orprevent, delay or inhibit metastasis, etc.

As used herein the term “therapeutically effective” applied to dose oramount refers to that quantity of a compound or pharmaceuticalcomposition that is sufficient to result in a desired activity uponadministration to a subject in need thereof. Within the context of thepresent invention, when the term “therapeutically effective” is used inconnection with a CASP1 inhibitor, it refers to an amount of said CASP1inhibitor or a pharmaceutical composition containing such CASP1inhibitor that is effective to sensitize cancer cells within the treatedsubject to glucocorticoid-induced apoptosis or cell death. When the term“therapeutically effective” is used in connection with a glucocorticoid,it refers to that quantity of the glucocorticoid or a pharmaceuticalcomposition containing such glucocorticoid that is sufficient to delaythe manifestation, arrest the progression, relieve or alleviate at leastone symptom of a disorder treated by the methods of the presentinvention. Note that when a combination of active ingredients isadministered (e.g., a combination of a CASP1 inhibitor and aglucocorticoid) the effective amount of the combination may or may notinclude amounts of each ingredient that would have been effective ifadministered individually.

The phrase “pharmaceutically acceptable”, as used in connection withcompositions of the invention, refers to molecular entities and otheringredients of such compositions that are physiologically tolerable anddo not typically produce untoward reactions when administered to amammal (e.g., a human). Preferably, as used herein, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in mammals, and moreparticularly in humans.

As used herein, the term “subject” refers to any mammal. In a preferredembodiment, the subject is human.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M J. Gait ed.1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higginseds.(1985»; Transcription and Translation (B. D. Hames & S. J. Higgins,eds. (1984»; Animal Cell Culture (R. I. Freshney, ed. (1986»;Immobilized Cells and Enzymes (lRL Press, (1986»; B. Perbal, A practicalGuide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (1994); amongothers.

Treatment/Therapeutic Methods of the Invention

In one embodiment, the invention provides a method for sensitizing acancer cell to glucocorticoid-induced apoptosis or cell death, whereinsaid cell is resistant to glucocorticoid-induced apoptosis or celldeath, comprising contacting the cell with an effective amount of aninhibitor of CASP1.

In another embodiment, the invention provides a method for killing acancer cell, wherein said cell is resistant to glucocorticoid-inducedapoptosis or cell death, comprising contacting the cell with an amountof an inhibitor of CASP1 that is effective to sensitize the cell toglucocorticoid-induced apoptosis or cell death and further comprisingcontacting the cell with an effective amount of a glucocorticoid.

In a further embodiment, the invention provides a method of treating aglucocorticoid-resistant cancer in a subject in need thereof whichmethod comprises administering to the subject (i) a therapeuticallyeffective amount of an inhibitor of CASP1 and (ii) a therapeuticallyeffective amount of a glucocorticoid. In one embodiment, thetherapeutically effective amount of the inhibitor of CASP1 is an amountthat is effective to sensitize cancer cells within the subject toglucocorticoid-induced apoptosis or cell death. The inhibitor of CASP1and the glucocorticoid can be administered simultaneously (in the samecomposition or in separate compositions) or they can be administeredsequentially (preferably, with the inhibitor of CASP1 being administeredprior to the glucocorticoid). In one specific embodiment, the treatmentmethod of the invention further comprises administering an additionalanti-cancer treatment to the subject.

Non-limiting examples of inhibitors of CASP1 useful in the methods ofthe invention are small molecules and antibodies, including inhibitorsof CASP1 protein function as well as inhibitors of CASP1 expression(such as, e.g., interfering RNA, dsRNA, RNA polymerase III transcribedDNAs, ribozymes, and antisense nucleic acids). Inhibitors of CASP1useful in the methods of the invention include (i) molecules whichdirectly inhibit expression or function of CASP1, (ii) molecules whichdirectly inhibit expression or function of NLRP3, and (iii) moleculeswhich inhibit NLRP3 inflammasome formation or NLRP3 inflammasomeactivity. A more detailed discussion of the inhibitors is providedbelow.

The inhibitors of CASP1 can be used in treatment/therapeutic methodsdescribed above or can be administered to a nonhuman mammal for thepurposes of obtaining preclinical data. Exemplary nonhuman mammals to betreated include nonhuman primates, dogs, cats, rodents and other mammalsin which preclinical studies are performed. Such mammals may beestablished animal models for a disease to be treated or may be used tostudy toxicity of the inhibitor of interest. In each of theseembodiments, dose escalation studies may be performed in the mammal.

The present invention can be used to treat any cancer which can betreated by a glucocorticoid and can acquire glucocorticoid resistanceaccompanied with increased expression of CASP1 and/or NLRP3.Non-limiting examples of cancers treatable by the methods of theinvention include, for example, carcinomas, lymphomas, sarcomas,blastomas, and leukemias. Non-limiting specific examples, include, forexample, acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia(AML), lymphoma, osteosarcoma, small-cell lung cancer, breast cancer,brain cancer, and multiple myeloma. Additional examples of cancerstreatable by the methods of the invention include, for example,pancreatic cancer, liver cancer, lung cancer, prostate cancer, coloncancer, renal cancer, bladder cancer, head and neck carcinoma, thyroidcarcinoma, soft tissue sarcoma, ovarian cancer, primary or metastaticmelanoma, squamous cell carcinoma, basal cell carcinoma, angiosarcoma,hemangiosarcoma, bone sarcoma, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, testicular cancer, uterine cancer, cervical cancer,gastrointestinal cancer, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, Waldenstroom's macroglobulinemia, papillaryadenocarcinomas, cystadenocarcinoma, bronchogenic carcinoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,lung carcinoma, epithelial carcinoma, cervical cancer, testicular tumor,glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, meningioma, retinoblastoma, leukemia, neuroblastoma,small cell lung carcinoma, bladder carcinoma, lymphoma, multiplemyeloma, medullary carcinoma, B cell lymphoma, T cell lymphoma, myeloma,leukemia, chronic myeloid leukemia, acute myeloid leukemia, chroniclymphocytic leukemia, acute lymphocytic leukemia, hematopoieticneoplasias, thymoma, sarcoma, non-Hodgkins lymphoma, Hodgkins lymphoma,uterine cancer, renal cell carcinoma, hepatoma, etc.

The methods of the present invention can be also applicable to otherdiseases (e.g., inflammatory and autoimmune diseases such as, e.g.,asthma) which are treated with glucocorticoids and can acquireglucocorticoid resistance accompanied with increased expression ofCASP1.

It is contemplated that when used to treat various diseases, CASP1inhibitors and glucocorticoids can be further combined with othertherapeutic agents suitable for treatment of such diseases. Also, two ormore CASP1 inhibitors and/or two or more glucocorticoids may beco-administered to generate additive or synergistic effects. Suitabletherapeutically effective dosages for each agent may be lowered due tothe additive action or synergy.

Therapeutic methods of the invention can be combined with additionalanti-cancer therapies such as, e.g., surgery, radiotherapy, chemotherapyor combinations thereof, depending on type of the tumor, patientcondition, other health issues, and a variety of factors. In certainaspects, other therapeutic agents useful for combination cancer therapywith the inhibitors of the invention include anti-angiogenic agents.Many anti-angiogenic agents have been identified and are known in theart, including, e.g., TNP-470, platelet factor 4, thrombospondin-1,tissue inhibitors of metalloproteases (TIMP1 and TIMP2), prolactin(16-Kd fragment), angiostatin (38-Kd fragment of plasminogen),endostatin, bFGF soluble receptor, transforming growth factor beta,interferon alpha, soluble KDR and FLT-1 receptors, placentalproliferin-related protein, VEGF antagonists, VEGF receptor antagonists(such as anti-VEGF antibodies), VEGF variants, soluble VEGF receptorfragments, aptamers capable of blocking VEGF or VEGFR, neutralizinganti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinases, and anycombinations thereof (e.g., anti-hVEGF antibody A4.6.1, bevacizumab orranibizumab). See also Carmeliet and Jain (2000).

Non-limiting examples of chemotherapeutic compounds which can be used incombination treatments of the present invention include, for example,aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg,bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine,carboplatin, carmustine, chlorambucil, cisplatin, cladribine,clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine,dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol,docetaxel, doxorubicin, epirubicin, estramnustine, etoposide,exemestane, filgrastim, fludarabine, fluorouracil, fluoxymesterone,flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin,ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole,leucovorin, leuprolide, levamisole, lomustine, mechlorethamine,melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane,mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,paclitaxel, pamidronate, pentostatin, plicamycin, porfimer,procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen,temozolomide, teniposide, thioguanine, thiotepa, titanocene dichloride,topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine,and vinorelbine.

These chemotherapeutic compounds may be categorized by their mechanismof action into, for example, following groups:anti-metabolites/anti-cancer agents, such as pyrimidine analogs(5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine)and purine analogs, folate antagonists and related inhibitors(mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine(cladribine)); antiproliferative/antimitotic agents including naturalproducts such as vinca alkaloids (vinblastine, vincristine, andvinorelbine), microtubule disruptors such as taxane (paclitaxel,docetaxel), vincristin, vinblastin, nocodazole, epothilones andnavelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damagingagents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan,camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide,cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin,hexamethyhnelamineoxaliplatin, iphosphamide, melphalan,merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramideand etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D),daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines,mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin;enzymes (L-asparaginase which systemically metabolizes L-asparagine anddeprives cells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,streptozocin), trazenes-dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,nilutamide) and aromatase inhibitors (letrozole, anastrozole);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, dipyridamole, ticlopidine,clopidogrel, abciximab; antimigratory agents; antisecretory agents(breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506),sirolimus (rapamycin), azathioprine, mycophenolate mofetil);anti-angiogenic compounds (e.g., TNP-470, genistein, bevacizumab) andgrowth factor inhibitors (e.g., fibroblast growth factor (FGF)inhibitors); angiotensin receptor blocker; nitric oxide donors;anti-sense oligonucleotides; antibodies (trastuzumab); cell cycleinhibitors and differentiation inducers (tretinoin); mTOR inhibitors,topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine,camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin,etoposide, idarubicin and mitoxantrone, topotecan, irinotecan); growthfactor signal transduction kinase inhibitors; mitochondrial dysfunctioninducers; and chromatin disruptors.

CASP1 Inhibitors of the Invention

The present invention encompasses various small molecule inhibitors ofCASP1 gene expression and/or CASP1 protein function. The presentinvention also encompasses various small molecule inhibitors of NLRP3gene expression and/or NLRP3 protein function.

Small molecules are a diverse group of synthetic and natural substancesgenerally having low molecular weights (preferably less than about 2000Daltons, less than about 1000 Daltons, or less than about 500 Daltons).Small molecules, without limitation, may be, for example, nucleic acids,peptides, polypeptides, peptide nucleic acids, peptidomimetics,carbohydrates, lipids, or other organic (carbon containing) or inorganicmolecules and may be synthetic or naturally occurring or optionallyderivatized. Such small molecules may be a therapeutically deliverablesubstance or may be further derivatized to facilitate delivery ortargeting.

Many CASP1 inhibitors useful in the methods of the present invention areknown in the art. Specific non-limiting examples of useful CASP1inhibitors include, e.g.,2-valyl-alanyl-3-amino-4-oxo-5-(2,6-dichlorobenzoyl)oxopentanoic acid(z-VAD-DCB), Ac-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-CHO) (SEQ ID NO: 18),Ac-YVAD-chloromethylketone (SEQ ID NO: 19), cytokine response modifier A(crmA), Pralnacasan (VX-740;(1S,9S)-N-[(2R,3S)-2-ethoxy-5-oxooxolan-3-yl]-9-(isoquinoline-1-carbonylamino)-6,10-dioxo-2,3,4,7,8,9-hexahydro-1H-pyridazino[1,2-a]diazepine-1-carboxamide), IDN-6556 (N-[2-(tert-butyl)phenyl]-2-oxo),VX-765((S)-1-((R)-2-(4-amino-3-chlorobenzamido)-3,3-dimethylbutanoyl)-N-((2S,3S)-2-ethoxy-5-oxotetrahydro-furan-3-yl)pyrrolidine-2-carboxamide),VRT-043198((S)-3-((S)-1-((R)-2-(4-amino-3-chlorobenzamido)-3,3-dimethylbutanoyl)pyrrolidine-2-carboxamido)-4-oxobutanoicacid), . and ML132 (CID-4462093; NCGC-00183434), Of these, the last fouragents are active site inhibitors that act through reversible(Pralnacasan and VX-765) or irreversible (IDN-6556) covalentmodification of the catalytic cysteine residue. See, e.g., Boxer et al.,ChemMedChem (2010), 5(5), 730-738; Eda, (2009), 251-287; Boxer et al., Asmall molecule inhibitor of Caspase 1. 2010 Feb. 25 [Updated 2011 Mar.3]. In: Probe Reports from the NIH Molecular Libraries Program[Internet]. Bethesda (Md.): National Center for BiotechnologyInformation (US); 2010-. Available from:http://www.ncbi.nlm.nih.gov/books/NBK56241/. Additional non-limitingexamples of useful inhibitors are provided, e.g., in the followingdocuments: International Patent Publ. Nos. WO 03/103599, WO 09/083929,WO 05/115362, WO 2011/094426, WO 04/058718, WO 04/002961, WO 03/088917,WO 03/068242, WO 03/042169, WO 98/16505, WO 93/09135, WO 03/106460, WO03/103677, WO 03/104231, WO 02/085899, WO 00/55114, WO 00/55127, WO00/61542, WO 01/05772, WO 01/10383, WO 01/16093, WO 01/42216, WO01/72707, WO 01/90070, WO 01/94351, WO 02/094263, WO 02/42278, WO99/47545, WO 01/90063, WO 02/22611, WO 02/12638, WO 95/35308, WO97/22619, WO 01/00658, WO 98/10778, WO 03/072528, WO 03/032918, WO05/003100, WO 04/002401, WO 00/61542, WO 00/55114, WO 99/47154, WO99/56765, WO 93/05071, WO 04/058718, WO 04/002961, WO 95/35308, WO97/22619, WO 99/47545, WO 01/90063, WO 95/35308, WO 97/22619, WO99/47545, WO 2002/089749, WO 99/36426, WO 98/16505, WO 98/16504, WO98/16502, and WO 01/90063; U.S. Pat. Nos. 6,184,210; 6,184,244;6,187,771; 6,197,750; 6,242,422; 5,716,929; 6,204,261; 7,115,654;6,693,096; 6,610,683; 6,531,467; 6,528,506; 6,200,969; 6,716,818;6,620,782; 6,566,338; 6,495,522; 6,355,618; 6,153,591; 6,083,981;5,932,549; 5,919,790; 5,744,451; 6,316,415; 5,932,549; 5,919,790;5,744,451; U.S. Patent Appl. Pub. Nos. 2002/0058630, 2004/0014753,2004/0009966, 2003/0236296, European Patent Nos. EP 1082127, EP 1049703,EP 0932600, EP 0932598, EP 0600880, and EP 1378573.

The above compounds may be obtained by methods known to skilledpractitioners and the methods disclosed in documents cited herein.

Additional CASP1 inhibitors can be isolated from natural sources (forexample, plants, fungi, microbes and the like) or isolated from randomor combinatorial chemical libraries of synthetic or natural compounds,or synthesized. See Werner et al., (2006) Brief Funct. Genomic Proteomic5(1):32-6. Many random or combinatorial libraries are known in the artthat can be used. Numerous means are currently used for random anddirected synthesis of saccharide, peptide, and nucleic acid basedcompounds. Synthetic compound libraries are commercially available fromMaybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton,N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (NewMilford, Conn.). A rare chemical library is available from Aldrich(Milwaukee, Wis.). Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available frome.g. Pan Laboratories (Bothell, Wash.) or MycoSearch (N.C.), or arereadily producible. Additionally, natural and synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical, and biochemical means (Blondelle et al., (1996) TibTech 14:60).

Methods for preparing libraries of molecules are well known in the artand many libraries are commercially available. Libraries of interest inthe invention include peptide libraries, randomized oligonucleotidelibraries, synthetic organic combinatorial libraries, and the like.Degenerate peptide libraries can be readily prepared in solution, inimmobilized form as bacterial flagella peptide display libraries or asphage display libraries. Peptide ligands can be selected fromcombinatorial libraries of peptides containing at least one amino acid.Libraries can be synthesized of peptoids and non-peptide syntheticmoieties. Such libraries can further be synthesized which containnon-peptide synthetic moieties, which are less subject to enzymaticdegradation compared to their naturally-occurring counterparts.Libraries are also meant to include for example but are not limited topeptide-on-plasmid libraries, polysome libraries, aptamer libraries,synthetic peptide libraries, synthetic small molecule libraries andchemical libraries. The libraries can also comprise cyclic carbon orheterocyclic structure and/or aromatic or polyaromatic structuressubstituted with one or more of the above-identified functional groups.

Examples of chemically synthesized libraries are described in Fodor etal., (1991) Science 251:767-773; Houghten et al., (1991) Nature354:84-86; Lam et al., (1991) Nature 354:82-84; Medynski, (1994)BioTechnology 12:709-710; Gallop et al., (1994) J. Medicinal Chemistry37(9):1233-1251; Ohlmeyer et al., (1993) Proc. Natl. Acad. Sci. USA90:10922-10926; Erb et al., (1994) Proc. Natl. Acad. Sci. USA91:11422-11426; Houghten et al., (1992) Biotechniques 13:412;Jayawickreme et al., (1994) Proc. Natl. Acad. Sci. USA 91:1614-1618;Salmon et al., (1993) Proc. Natl. Acad. Sci. USA 90:11708-11712; PCTPublication No. WO 93/20242, dated Oct. 14, 1993; and Brenner et al.,(1992) Proc. Natl. Acad. Sci. USA 89:5381-5383.

Examples of phage display libraries are described in Scott et al.,(1990) Science 249:386-390; Devlin et al., (1990) Science, 249:404-406;Christian, et al., (1992) J. Mol. Biol. 227:711-718; Lenstra, (1992) J.Immunol. Meth. 152:149-157; Kay et al., (1993) Gene 128:59-65; and PCTPublication No. WO 94/18318.

Screening the libraries can be accomplished by any variety of commonlyknown methods. See, for example, the following references, whichdisclose screening of peptide libraries: Parmley and Smith, (1989) Adv.Exp. Med. Biol. 251:215-218; Scott and Smith, (1990) Science249:386-390; Fowlkes et al., (1992) BioTechniques 13:422-427; Oldenburget al., (1992) Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al.,(1994) Cell 76:933-945; Staudt et al., (1988) Science 241:577-580; Bocket al., (1992) Nature 355:564-566; Tuerk et al., (1992) Proc. Natl.Acad. Sci. USA 89:6988-6992; Ellington et al., (1992) Nature355:850-852; U.S. Pat. Nos. 5,096,815; 5,223,409; and 5,198,346, all toLadner et al.; Rebar et al., (1993) Science 263:671-673; and PCT Pub. WO94/18318.

Identification and screening of CASP1 inhibitors can be furtherfacilitated by X-ray crystallography, neutron diffraction, nuclearmagnetic resonance spectrometry, and other techniques for structuredetermination. These techniques provide for the rational design oridentification of inhibitors.

The present invention also encompasses inhibitors of CASP1 geneexpression, including inhibitors of CASP1 protein production, as well asinhibitors of NLRP3 gene expression, including inhibitors of NLRP3protein production. Non-limiting examples of such useful expressioninhibitors include, e.g., interfering RNA (e.g., siRNA or shRNA), dsRNA,RNA polymerase III transcribed DNAs, ribozymes, and antisense nucleicacids.

Antisense oligonucleotides, including antisense DNA, RNA, and DNA/RNAmolecules, act to directly block the translation of mRNA by binding totargeted mRNA and preventing protein translation. For example, antisenseoligonucleotides of at least about 15 bases and complementary to uniqueregions of the target DNA sequence can be synthesized, e.g., byconventional phosphodiester techniques (Dallas et al., (2006) Med. Sci.Monit. 12(4):RA67-74; Kalota et al., (2006) Handb. Exp. Pharmacol.173:173-96; Lutzelburger et al., (2006) Handb. Exp. Pharmacol.173:243-59).

siRNA comprises a double stranded structure typically containing 15 to50 base pairs and preferably 21 to 25 base pairs and having a nucleotidesequence identical or nearly identical to an expressed target gene orRNA within the cell. Antisense polynucleotides include, but are notlimited to: morpholinos, 2′-O-methyl polynucleotides, DNA, RNA and thelike.

RNA polymerase III transcribed DNAs contain promoters, such as the U6promoter. These DNAs can be transcribed to produce small hairpin RNAs inthe cell that can function as siRNA or linear RNAs that can function asantisense RNA. The inhibitor may be polymerized in vitro, recombinantRNA, contain chimeric sequences, or derivatives of these groups. Theinhibitor may contain ribonucleotides, deoxyribonucleotides, syntheticnucleotides, or any suitable combination such that the target RNA and/orgene is inhibited. In addition, these forms of nucleic acid may besingle, double, triple, or quadruple stranded. (see for example Bass(2001) Nature, 411, 428 429; Elbashir et al., (2001) Nature, 411, 494498; and PCT Publication Nos. WO 00/44895, WO 01/36646, WO 99/32619, WO00/01846, WO 01/29058, WO 99/07409, WO 00/44914).

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. The mechanism of ribozyme action involves sequencespecific hybridization of the ribozyme molecule to complementary targetRNA, followed by endonucleolytic cleavage. Engineered hammerhead motifribozyme molecules that specifically and efficiently catalyzeendonucleolytic cleavage of mRNA sequences are also within the scope ofthe present invention. Scanning the target molecules for ribozymecleavage sites that include the following sequences, GUA, GUU, and GUCinitially identifies specific ribozyme cleavage sites within anypotential RNA target. Once identified, short RNA sequences of betweenabout 15 and 20 ribonucleotides corresponding to the region of thetarget gene containing the cleavage site can be evaluated for predictedstructural features such as secondary structure that may render theoligonucleotide sequence unsuitable. The suitability of candidatetargets can also be evaluated by testing their accessibility tohybridization with complementary oligonucleotides using, e.g.,ribonuclease protection assays.

Expression inhibitors of the present invention can be prepared by knownmethods. These include techniques for chemical synthesis such as, e.g.,by solid phase phosphoamite chemical synthesis. Alternatively, antisenseRNA molecules can be generated by in vitro or in vivo transcription ofDNA sequences encoding the RNA molecule. Such DNA sequences can beincorporated into a wide variety of vectors that incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.See, e.g., Weintraub, H. et al., Antisense RNA as a molecular tool forgenetic analysis, Reviews—Trends in Genetics, Vol. 1 (1) 1986.

Various modifications to the oligonucleotides can be introduced as ameans of increasing intracellular stability and half-life. Possiblemodifications include but are not limited to the addition of flankingsequences of ribonucleotides or deoxyribonucleotides to the 5′ and/or 3′ends of the molecule, or the use of phosphorothioate or 2′-O-methylrather than phosphodiesterase linkages within the oligonucleotidebackbone.

Aptamers nucleic acid sequences are readily made that bind to a widevariety of target molecules. The aptamer nucleic acid sequences usefulin the methods of the invention can be comprised entirely of RNA orpartially of RNA, or entirely or partially of DNA and/or othernucleotide analogs. Aptamers are typically developed to bind particularligands by employing known in vivo or in vitro (most typically, invitro) selection techniques known as SELEX (Systematic Evolution ofLigands by Exponential Enrichment). Methods of making aptamers aredescribed in, for example, Ellington and Szostak (1990) Nature 346:818,Tuerk and Gold (1990) Science 249:505, U.S. Pat. No. 5,582,981; PCTPublication No. WO 00/20040; U.S. Pat. No. 5,270,163; Lorsch and Szostak(1994) Biochem. 33:973; Mannironi et al., (1997) Biochem. 36:9726; Blind(1999) Proc. Nat'l. Acad. Sci. USA 96:3606-3610; Huizenga and Szostak(1995) Biochem. 34:656-665; PCT Publication Nos. WO 99/54506, WO99/27133, and WO 97/42317; and U.S. Pat. No. 5,756,291.

Another class of CASP1 inhibitors useful in the methods of the inventionare inhibitory antibodies. The antibodies for use in accordance with thepresent invention may be monoclonal or polyclonal as appropriate. Theantibody fragments can be also used and include, for example, Fab, Fab′,F(ab′)₂ or Fv fragments. The antibody may be a single chain antibody.Other suitable modifications and/or agents will be apparent to thoseskilled in the art. Chimeric and humanized antibodies are also withinthe scope of the invention. It is expected that chimeric and humanizedantibodies would be less immunogenic in a human subject than thecorresponding non-chimeric antibody. A variety of approaches for makingchimeric antibodies, comprising for example a non-human variable regionand a human constant region, have been described. See, for example,Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81,6851 (1985); Takeda,et al., Nature 314,452 (1985), Cabilly et al., U.S. Pat. No. 4,816,567;Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., European PatentPublication EP 171496; European Patent Publication 0173494, UnitedKingdom Patent GB 2177096B. Additionally, a chimeric antibody can befurther “humanized” such that parts of the variable regions, especiallythe conserved framework regions of the antigen-binding domain, are ofhuman origin and only the hypervariable regions are of non-human origin.Such altered immunoglobulin molecules may be made by any of severaltechniques known in the art, (e.g., Teng et al., Proc. Natl. Acad. Sci.U.S.A., 80, 7308-7312 (1983); Kozbor et al., Immunology Today, 4, 7279(1983); Olsson et al., Meth. Enzymol., 92, 3-16 (1982)), and arepreferably made according to the teachings of PCT Publication WO92/06193or EP 0239400. Humanized antibodies can be commercially produced by, forexample, Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, GreatBritain.

In certain embodiments, anti-idiotypic antibodies can be also used.Anti-idiotypic antibodies recognize antigenic determinants associatedwith the antigen-binding site of another antibody. Anti-idiotypicantibodies can be prepared against a second antibody by immunizing ananimal of the same species, and preferably of the same strain, as theanimal used to produce the second antibody. See, e.g., U.S. Pat. No.4,699,880. In one embodiment, antibodies are raised against CASP1 or aportion thereof, and these antibodies are used in turn to produce ananti-idiotypic antibody.

Preferably, the CASP1 inhibitory antibodies useful in the methods of theinvention provide intracellular targeting. Intracellular targeting canbe accomplished, for example, through the use of intracellularlyexpressed antibodies referred to as intrabodies.

To screen for antibodies which bind to a particular epitope on theantigen of interest (e.g., CASP1), a routine cross-blocking assay suchas that described in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping, e.g. as described in Champe et al.(1995) J. Biol. Chem. 270:1388-1394, can be performed to determinewhether the antibody binds an epitope of interest.

Additional antibodies useful in the methods of the present invention canbe also generated and selected using phage display approach asdescribed, e.g., in U.S. Patent Appl. Publ. No. 2008/0213268.

Antibodies can be further modified to generate antibody mutants withimproved physical, chemical and or biological properties over the parentantibody. See, e.g., Amit et al. (1986) Science 233:747-753; Chothia etal. (1987) J. Mol. Biol. 196:901-917; EP 239400B; Cunningham and Wells(1989) Science 244:1081-1085.

Antibodies can be prepared by standard means. See, e.g., Kohler et al.,Nature 256:495-497 (1975) and Eur. J. Immunol. 6:511-519 (1976);Milstein et al., Nature 266:550-552 (1977); Koprowski et al., U.S. Pat.No. 4,172,124; Harlow and Lane, “Antibodies: A Laboratory Manual,” (ColdSpring Harbor Laboratory: Cold Spring Harbor, N.Y., 1988); and “CurrentProtocols In Molecular Biology,” (Ausubel et al., Eds.; John Wiley &Sons: New York, N.Y., 1991); Kozbar et al., Immunology Today 4:72(1983)), Cole et al., “Monoclonal Antibodies and Cancer Therapy” (AlanR. Liss, Inc. pp. 77-96 (1985)); Cabilly et al., U.S. Pat. No.4,816,567; Winter, U.S. Pat. No. 5,225,539; Cabilly et al., EuropeanPatent No. 0,125,023; Boss et al., U.S. Pat. No. 4,816,397; Boss et al.,European Patent No.0,120,694; Neuberger et al., WO 86/01533; Neubergeret al., European Patent No. 0,194,276; Winter, European Patent No.0,239,400; Newman et al., BioTechnology 10:1455-1460 (1992); Ladner etal., U.S. Pat. No. 4,946,778; Bird et al., Science 242:423-426 (1988);Kamman et al., Nucl. Acids Res., 17:5404 (1989)); Sato et al., CancerResearch 53:851-856 (1993); Daugherty et al., Nucleic Acids Res.19(9):2471-2476 (1991); Lewis and Crowe, Gene 101:297-302 (1991);Krebber et al., U.S. Pat. No. 5,514,548; and Hoogenboom et al., WO93/06213; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551-2555(1993); Jakobovits et al., Nature 362:255-258 (1993); Lonberg et al.,U.S. Pat. No. 5,545,806; Surani et al., U.S. Pat. No. 5,545,807; Queenet al., European Patent No. 0,451,216; Boss et al., U.S. Pat. No.4,816,397; Boss et al., European Patent No. 0,120,694; Neuberger et al.,WO 86/01533; Padlan et al., European Patent Application No. 0,519,596;Ladner et al., U.S. Pat. No. 4,946,778; and Huston, U.S. Pat. No.5,476,786.

“Upstream” Inhibitors of CASP1

In addition to inhibitors which directly affect expression of CASP1 geneand/or function of CASP1 protein and inhibitors which directly affectexpression of NLRP3 gene and/or function of NLRP3 protein, inhibitorswhich are useful in the methods of the invention include inhibitors ofNLRP3 inflammasome formation and inhibitors of NLRP3 inflammasomeactivity. Non-limiting examples of inhibitors of NLRP3 inflammasomeformation and/or NLRP3 inflammasome activity useful in the methods ofthe invention include, e.g., small molecular weight compounds fitting inthe ATP binding site of NLRP3 and nitric oxide (NO) donors (e.g.,S-nitroso-N-acetylpenicillamine (SNAP)). See, e.g., Hernandez-Cuellar etal., J Immunol., 2012, 189(11):5113-5117. Other useful inhibitorsinclude small molecular weight compounds specifically blocking HSP90,such as geldanamycin, or less toxic derivates, such as 17-AAG(17-(Allylamino)-17-demethoxygeldanamycin) or 17-DMAG(17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin) and theirpharmaceutically acceptable salts, have been successfully developed.These inhibitors are effective in the HSP90-dependent assembly of theNLRP3 inflammasome. See, e.g., U.S. Patent Appl. Pub. No. 2011/0262449.Other geldanamycin derivatives are known in the art such as compoundsdisclosed in U.S. Pat. No. 4,261,989, US 2004/0235813, WO 02/36574, WO02/079167, WO 03/02671 and WO 2005/095347. Other inhibitors of HSP90 arealso known in the art, such as compounds disclosed in WO 2006/095783, WO2006/092202, WO 2006/090094, WO 2006/087077, WO 2006/084030, WO2005/028434, WO 2004/072051, WO 2006/079789, US 2006-0167070, WO2006/075095, US 2006-0148817, WO 2006/057396, WO 2006/055760, WO02/069900, WO 2006/052795, WO 2006/050373, WO 2006/051808, WO2006/039977, US 2006/0073151, EP 1 642 880, EP 1 631 267, EP 1 628 667,US 2006/0035837, WO 2006/008503, WO 2006/010595, WO 2006/010594, WO2006/003384, WO 2005/115431, EP 1 620 090, WO 2005/061461, WO2005/063222, US 2005/0049263, WO 2004/050087, WO 2004/024142, WO2004/024141, WO 03/067262, WO 03/055860, WO 03/041643, WO 03/037860.

Methods for Administering Compositions Comprising CASP1 Inhibitors

The appropriate dosages of CASP1 inhibitors used in the methods of theinvention will depend on the type of disease to be treated, the severityand course of the disease, previous therapy, the patient's clinicalhistory and response to the inhibitor, and the discretion of theattending physician. A CASP1 inhibitor can be administered to thepatient at one time or over a series of treatments. The progress of thetherapy of the invention can be easily monitored by conventionaltechniques and assays.

The administration of CASP1 inhibitors according to the methods of theinvention can be performed by any suitable route, including systemicadministration as well as administration directly to the site of thedisease (e.g., to primary tumor).

In certain embodiments, the CASP1 inhibitors are formulated inpharmaceutical compositions with a pharmaceutically acceptable carrieror excipient.

In a separate embodiment, the invention also provides pharmaceuticalcompositions comprising a CASP1 inhibitor, a glucocorticoid, and apharmaceutically acceptable carrier or excipient.

The formulations used in the methods of the invention may convenientlybe presented in unit dosage form and may be prepared by methods known inthe art. The amount of active ingredients that can be combined with acarrier material to produce a single dosage form will vary dependingupon the host being treated and the particular mode of administration.The amount of active ingredients that can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the compound which produces a therapeutic effect.

In general, the formulations can be prepared with a liquid carrier, or afinely divided solid carrier, or both, and then, if necessary, shapingthe product.

Formulations for oral administration may be in the form of capsules,cachets, pills, tablets, powders, granules, or as a solution or asuspension in an aqueous or non-aqueous liquid, or as anoil-in-water orwater-in-oil liquid emulsion, and the like, each containing apredetermined amount of one or more active ingredients.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more activeingredients can be mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

Suspensions, in addition to one or more active ingredients, can containsuspending agents such as ethoxylated isostearyl alcohols,polyoxyethylene sorbitol, and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Compositions of the invention can be also administered topically, eitherto skin or to mucosal membranes. The topical formulations may furtherinclude one or more of the wide variety of agents known to be effectiveas skin or stratum corneum penetration enhancers. Examples of these are2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide,dimethylformamide, propylene glycol, methyl or isopropyl alcohol,dimethyl sulfoxide, and azone. Additional agents may further be includedto make the formulation cosmetically acceptable. Examples of these arefats, waxes, oils, dyes, fragrances, preservatives, stabilizers, andsurface active agents. Keratolytic agents such as those known in the artmay also be included. Examples are salicylic acid and sulfur.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, and inhalants. The subject therapeutic agents may be mixedunder sterile conditions with a pharmaceutically acceptable carrier, andwith any preservatives, buffers, or propellants which may be required.The ointments, pastes, creams and gels may contain, in addition to asubject polypeptide agent, excipients, such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to one or more activeingredients, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Pharmaceutical compositions suitable for parenteral administration maycomprise one or more active ingredients in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions can also contain preservatives, wetting agents,emulsifying agents and dispersing agents. Prevention of the action ofmicroorganisms may be ensured by the inclusion of various antibacterialand antifungal agents, for example, paraben, chlorobutanol, phenolsorbic acid, and the like. It may also be desirable to include isotonicagents, such as sugars, sodium chloride, and the like into thecompositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption, such as aluminum monostearate and gelatin.

Injectable depot forms can be made by forming microencapsule matrices ofone or more active ingredients in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of active ingredientto polymer, and the nature of the particular polymer employed, the rateof antagonist release can be controlled. Examples of other biodegradablepolymers include poly(orthoesters) and poly(anhydrides). Depotinjectable formulations are also prepared by entrapping the antagonistsin liposomes or microemulsions which are compatible with body tissue.

Formulations for intravaginal or rectal administration may be presentedas a suppository, which may be prepared by mixing one or more activeingredients with one or more suitable nonirritating excipients orcarriers comprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Diagnostic Methods of the Invention

In one embodiment, the invention provides a method for determiningwhether a cancer in a subject is likely to be resistant to aglucocorticoid treatment, said method comprising the steps of:(d) determining the expression level of CASP1 gene and/or NLRP3 gene incancer cells from the subject;(e) comparing the expression level of each gene determined in step (a)with a corresponding control expression level for that gene, and(f) (i) identifying that the cancer in the subject is likely to beresistant to the glucocorticoid treatment when the expression level ofCASP1 gene and/or NLRP3 gene in step (a) is increased by at least 1.5fold as compared to the corresponding control expression level or (ii)identifying that the cancer in the subject is not likely to be resistantto the glucocorticoid treatment when the expression level of CASP1 geneand/or NLRP3 gene in step (a) is not increased or is increased by lessthan 1.5 fold as compared to the corresponding control expression level.

In one specific embodiment, the corresponding control expression levelis the expression level of the same gene in similarly processed cancercells of the same type which are sensitive to glucocorticoid treatment.In another specific embodiment, the corresponding control expressionlevel is a predetermined standard.

For determination of CASP1 or NLRP3 expression levels, RNA can beextracted from the collected cancer cells or tumor samples and purifiedusing a variety of standard procedures as described, for example, in RNAMethodologies, A laboratory guide for isolation and characterization,2nd edition, 1998, Robert E. Farrell, Jr., Ed., Academic Press. Inaddition, various commercial products are available for RNA isolation.

The expression levels of CASP1 or NLRP3 can be determined using any ofvarious techniques known in the art. Such methods include, withoutlimitation, amplification-based assays such as RT-PCR (e.g., TAQMAN),hybridization-based assays such as DNA microarray analysis,flap-endonuclease-based assays (e.g., INVADER), and direct mRNA capture(QUANTIGENE or HYBRID CAPTURE (Digene)). See, for example, US2010/0190173 for descriptions of representative methods that can be usedto determine expression levels.

Methods of detecting and/or quantifying the level of CASP1 and/or NLRP3gene transcripts (mRNA or cDNA made therefrom) using nucleic acidhybridization techniques are known to those of skill in the art.Non-limiting examples of useful hybridization techniques includeNorthern blotting, dot blotting, in situ hybridization, RNaseprotection, probing DNA microchip arrays, and the like.

In another embodiment, amplification-based assays are used to measurethe expression level of CASP1 and/or NLRP3. In such assays, the CASP1and/or NLRP3 nucleic acid sequences act as a template in anamplification reaction (e.g., PCR). In a quantitative amplification, theamount of amplification product will be proportional to the amount oftemplate in the original sample. Comparison to appropriate controlsprovides a measure of the level of CASP1 and/or NLRP3 in the sample.Methods of quantitative amplification are well known to those of skillin the art. Detailed protocols for quantitative PCR are provided, e.g.,in Innis et al. (1990) PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc. N.Y.). The known nucleic acidsequences for CASP1 and/or NLRP3 enable one of skill to routinely selectprimers to amplify any portion of these genes. In one embodiment, aTaqMan based assay is used to quantify the CASP1 and/or NLRP3expression. TaqMan based assays use a fluorogenic oligonucleotide probethat contains a 5′ fluorescent dye and a 3′ quenching agent. The probehybridizes to a PCR product, but cannot itself be extended due to ablocking agent at the 3′ end. When the PCR product is amplified insubsequent cycles, the 5′ nuclease activity of the polymerase, e.g.,AmpliTaq, results in the cleavage of the TaqMan probe. This cleavageseparates the 5′ fluorescent dye and the 3′ quenching agent, therebyresulting in an increase in fluorescence as a function of amplification(see, for example, literature provided by Perkin-Elmer, e.g.,www2.perkin-elmer.com). Other suitable amplification methods include,but are not limited to, ligase chain reaction (LCR) (see, Wu and Wallace(1989) Genomics 4:560, Landegren et al. (1988) Science 241: 1077, andBarringer et al. (1990) Gene 89:117), transcription amplification (Kwoket al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), self-sustainedsequence replication (Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA87: 1874), dot PCR, and linker adapter PCR, etc.

In a separate embodiment, the expression level of CASP1 gene and/orNLRP3 gene is determined by determining the level of CASP1 and/or NLRP3protein (using e.g., antibodies).

In a separate embodiment, the invention provides a method fordetermining whether a cancer in a subject is likely to be resistant to aglucocorticoid treatment, said method comprising the steps of:

(d) determining the methylation level of CASP1 gene promoter and/orNLRP3 gene promoter in cancer cells from the subject;(e) comparing the methylation level of each gene promoter determined instep (a) with a corresponding control methylation level for that genepromoter, and(f) (i) identifying that the cancer in the subject is likely to beresistant to glucocorticoid treatment when the methylation level ofCASP1 gene promoter and/or NLRP3 gene promoter in step (a) is decreasedby at least 1.5 fold as compared to the corresponding controlmethylation level or (ii) identifying that the cancer in the subject isnot likely to be resistant to glucocorticoid treatment when themethylation level of CASP1 gene promoter and/or NLRP3 gene promoter instep (a) is not increased or is increased by less than 1.5 fold ascompared to the corresponding control methylation level.

In one specific embodiment, the CASP1 gene promoter comprises thesequence

(SEQ ID NO: 3) GTCGGGGAAGGTTTTGAGAAAGAAGGGTCCCTGGACAAGAACCTTGTCATTTTCTGAGTGGCCGGTACCGAAAAGAGAGGAGGGAAGAACACACTGACTTTGACTTTCATACGAAGCGGAAG

In one specific embodiment, the NLRP3 gene promoter comprises thesequence

(SEQ ID NO: 4) CTCCTTTGACTTCAACTCCTTATCACTTCTCAAACAGGTTACAGTATCGGGGCATTAGTTGCCCTGTTTTTAAAAGAACGACTACCCAGTTCTACCGTAGCACTTCACCAACAAGTGGCATT

In one specific embodiment, the corresponding control methylation levelis the methylation level of the same gene promoter in similarlyprocessed cancer cells of the same type which are sensitive toglucocorticoid treatment. In one specific embodiment, the correspondingcontrol methylation level is a predetermined standard.

Promoter methylation of CASP1 gene and/or NLRP3 gene can be determinedby any method known in the art. One example of a useful method isSouthern blot analysis after cleavage with methylation-sensitiverestriction endonucleases or PCR across the restriction sites of suchenzymes. Any restriction endonuclease that includes CG as part of itsrecognition site and that is inhibited when the C is methylated, can beutilized for this analysis. Methylation sensitive restrictionendonucleases include, for example, AciI, BsiEI, BssHII, BstUI, Eag I,FauI, HaeII, HpaI, HpaII, MspI, NarI, NotI, SacII, and SmaI. Theseenzymes may be used alone or in combination.

Another useful method for determining promoter methylation of CASP1 geneand/or NLRP3 gene is bisulfite DNA sequencing which is based on sodiumbisulfite-induced modification of genomic DNA under conditions wherebyunmethylated cytosine is converted to uracil. The bisulfite-modifiedsequence is then amplified by PCR with two sets of strand-specificprimers to yield a pair of fragments, one from each strand, in which alluracil and thymine residues are amplified as thymine and only5-methylcytosine residues are amplified as cytosine. The PCR productscan be sequenced directly or can be cloned and sequenced to providemethylation maps of single DNA molecules (see, e.g., Frommer, et al.,Proc. Natl. Acad. Sci. 89: 1827-1831, 1992). PCR can be either CD-PCRwith primer pairs located outside the CpG sites or MS-PCR with primersspecific for methylated (M primer) versus unmethylated (U primer) DNA(Herman et al., 1996). The difference between CD-PCR and MS-PCR is thelocation of primer site. In CD-PCR, primers are designed from theregions outside the CpG islands and the methylation status of the CpGislands is investigated from cloning and sequencing of the PCR product.In MS-PCR, primers are chosen from the CpG sites to discriminate betweenmethylated and unmethylated alleles following bisulfate treatment andthus the methylation status of the CpG islands is assessed directly fromthe PCR product. The PCR product is expected only in samples wheremethylated DNA is mixed with the M primer and unmethylated DNA is mixedwith U primer.

Other useful methods for determining promoter methylation of CASP1 geneand/or NLRP3 gene include Restriction Landmark Genome Scanning (RLGS)and Methylation-Sensitive Representational Difference Analysis (MS-RDA).In RLGS methylation changes are visualized as a dense cluster of “spots”on 2-dimensional gels. MS-RDA is a PCR-based technique that is biasedtoward short DNA fragments and against GC-rich sequences. Novelarray-based hybridization methods have also been developed. Anotheruseful method is Methylation Subtraction Analysis (MSA) which relies onthe enzymatic fractionation of the human genome into its methylated andunmethylated compartments.

In the diagnostic methods of the invention, after determining theexpression levels or promoter methylation levels of CASP1 and/or NLRP3,the patient's cancer can be classified as being likely or unlikely to beresistant to glucocorticoid therapy. The classification may bedetermined computationally based upon known methods in the art. Theresult of the computation may be displayed on a computer screen orpresented in a tangible form, for example, as a probability (e.g., from0 to 100%) of the patient. The report will aid a physician indetermining further treatment of the patient. For example, in certainembodiments, where the patient's expression levels or promotermethylation levels of CASP1 and/or NLRP3 will be prognostic ofresistance to glucocorticoid therapy, the patient will be subsequentlytreated with either (i) a combination of a CASP1 inhibitor and aglucocorticoid, or (ii) with increased (e.g., at least 1.5 fold morethan typical) doses of a glucocorticoid, or (iii) with anon-glucocorticoid chemotherapeutic and/or radiation. In certainembodiments, where the patient's expression levels or promotermethylation levels of CASP1 and/or NLRP3 will be prognostic of lack ofresistance to glucocorticoid therapy, the patient will be subsequentlytreated with a glucocorticoid.

In conjunction with the diagnostic methods of the invention, providedherein are various kits for diagnosing likelihood of cancer resistanceto glucocorticoid treatment. In one embodiment, such kit comprises oneor more pairs of oligonucleotides directed toward CASP1 and/or NLRP3,wherein said pairs of oligonucleotides can be used to determine theexpression levels of CASP1 gene and/or NLRP3 gene. The oligonucleotidesmay be designed to detect expression levels in accordance with any assayformat, including but not limited to those described herein. Such kitcan further comprise a detection means and/or an amplification meansand/or one or more pairs of control oligonucleotides. In anotherembodiment, a kit for diagnosing likelihood of cancer resistance toglucocorticoid treatment comprises antibodies directed toward CASP1protein and/or NLRP3 protein, wherein said antibodies can be used todetermine the expression levels of CASP1 and/or NLRP3 protein. Such kitcan further comprise a detection means and/or one or more controlantibodies.

In yet another embodiment, a kit for diagnosing likelihood of cancerresistance to glucocorticoid treatment comprises means for detectingpromoter methylation of CASP1 gene and/or NLRP3 gene. In one specificembodiment, such kit includes sodium bisulfite, hydroquinone andstandard components for DNA sequence determination either via Sangersequencing, including location specific oligonucleotide primers or vianext generation sequencing, or via solution bead based or solidsubstrate hybridization methods.

Any of the kits of the invention can optionally further compriseinstructions for use.

EXAMPLES

The present invention is also described and demonstrated by way of thefollowing examples. However, the use of these and other examplesanywhere in the specification is illustrative only and in no way limitsthe scope and meaning of the invention or of any exemplified term.Likewise, the invention is not limited to any particular preferredembodiments described here. Indeed, many modifications and variations ofthe invention may be apparent to those skilled in the art upon readingthis specification, and such variations can be made without departingfrom the invention in spirit or in scope. The invention is therefore tobe limited only by the terms of the appended claims along with the fullscope of equivalents to which those claims are entitled.

Example 1 Materials and Methods Patient Samples

Written informed assent or consent was obtained from patients or theirparents/guardians (as appropriate). The research and use of thesesamples were approved by the institutional review board at the hostinstitute.

Gene Expression Analyses

Total RNA was extracted with TriReagent (Molecular Research Center,Inc., Cincinnati, OH) from cryopreserved mononuclear cell suspensionsfrom patient bone marrow aspirates obtained at diagnosis. All geneexpression microarrays were performed by the St. Jude Children'sResearch Hospital, Hartwell Center for Bioinformatics & Biotechnology.High-quality RNA was hybridized to the HG-U133A (GPL96) or HG-U133 Plus2.0 (GPL570) oligonucleotide microarrays in accordance with themanufacturer's protocol (Affymetrix, Santa Clara, Calif.). Thesemicroarrays contain 22,283 or 54,675 gene probe sets, representingapproximately 18,400 or 47,400 human transcripts, respectively. Geneexpression data were MASS (Hubbell et al., 2002) processed using theaffy (Gautier et al., 2004) Bioconductor (Gentleman et al., 2004)R-project package or using Affymetrix Microarray Suite version 5.0(Cheok et al., 2003; Yeoh et al., 2002) as previously described(Holleman et al., 2004).

DNA Methylation Analyses

DNA was isolated at the time of diagnosis from lymphoblasts from patientbone marrow aspirates. Genome-wide DNA methylation status was determinedusing either an Infinium HumanMethylation27 BeadChip Kit or InfiniumHumanMethylation450 BeadChip Kit in accordance with the manufacturer'sprotocol (Illumina, San Diego, Calif.). HumanMethylation27 BeadChipswere performed at either Emory Integrated Genomics Core (EIGC) orWellcome Trust Centre for Human Genetics Genomics Lab, Oxford, UK.HumanMethylation450 BeadChip experiments were performed at the HeflinCenter for Genomic Science at the University of Alabama at Birmingham.DNA methylation status was classified as low if less than or equal to0.25 and high if greater than 0.25.

Statistical Analyses

Analyses were performed using R software (http://www.r-project.org)unless otherwise specified. Exact Wilcoxon Mann-Whitney Rank Sum testswere used for analyses of differential gene expression and differentialDNA methylation and Stouffer's Z-score method was used for meta-analysis(Stouffer et al., 1949). K-means clustering analysis with a k=2 was usedfor DNA methylation data and either untransformed or log-transformedgene expression data, with Fisher's exact test for determiningclustering significance. Fisher's exact test was used to assess thesignificance of enrichment of known glucocorticoid response elements.

CASP1 Enzymatic Assays

Recombinant human CASP1 (100-200 U, where U=1 pmol/min at 30° C., 200 μMYVAD-pNA (SEQ ID NO: 21)) from a CASP1 assay kit for drug discovery(Enzo Life Sciences, Farmingdale, N.Y., catalog number BML-AK701-0001)was incubated at 30° C. with wild-type or mutated GR in the presence (10μM unless otherwise indicated) or absence of inhibitors, (Ac-YVAD-CHO(SEQ ID NO: 18), Enzo Life Sciences, Farmingdale, N.Y., catalog numberBML-P403-9090), VX765, VRT-043198). Substrates for enzyme assays wereprepared in CASP1 assay buffer (Enzo Life Sciences, Farmingdale, N.Y.catalog number KI-111) consisting of 50 mM HEPES, pH7.4, 100 mM NaCl,0.1% CHAPS, 10 mM DTT, 1 mM EDTA and 10% glycerol.

Site-Directed Mutagenesis

Site-directed mutagenesis was performed on NR3C1 using a QuikChangeSite-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.).Mutagenesis of the LLID motif (SEQ ID NO: 20) was performed in 4sequential mutagenesis reactions from Myc-DDK tagged NR3C1 (Origene,Rockville, Md., catalog #RC220189). The following mutagenesis primerswere used:

MutF1-NR3C1: (SEQ ID NO: 5)CCTTGGAGATCAGACCTGTTGATAGCTGAAAACTGTTTGCTTTC; MutR1-NR3C1:(SEQ ID NO: 6) GAAAGCAAACAGTTTTCAGCTATCAACAGGTCTGATCTCCAAGG;MutF2-NR3C1: (SEQ ID NO: 7)CCTTGGAGATCAGACCTGTTGGCAGCTGAAAACTGTTTGCTTTC; MutR2-NR3C1:(SEQ ID NO: 8) GAAAGCAAACAGTTTTCAGCTGCCAACAGGTCTGATCTCCAAGG;MutF3-NR3C1: (SEQ ID NO: 9)CCTTGGAGATCAGACCTGGCGGCAGCTGAAAACTGTTTGCTTTC; MutR3-NR3C1:(SEQ ID NO: 10) GAAAGCAAACAGTTTTCAGCTGCCGCCAGGTCTGATCTCCAAGG;MutF4-NR3C1: (SEQ ID NO: 11)CCTTGGAGATCAGACGCGGCGGCAGCTGAAAACTGTTTGCTTTC; MutR4-NR3C1:(SEQ ID NO: 12) GAAAGCAAACAGTTTTCAGCTGCCGCCGCGTCTGATCTCCAAGG.

Successful mutagenesis was confirmed by Sanger sequencing.

Ex Vivo Drug Sensitivity Assays

Leukemia cells were isolated at diagnosis from patient bone marrowaspirates. If the leukemia cell percentage from diagnostic bone marrowsamples was less than ninety percent, magnetic activated cell sorting(Miltenyl Biotec, Auburn, Calif.) was performed to further enrich forleukemia cells. If red cell contamination was greater than thirtypercent, red blood cell lysis was performed. Cells were centrifuged at300 g for five minutes and resuspended at a concentration of two millioncells per milliliter. Eighty microliters of this leukemia cellsuspension was then plated into each experimental well of round-bottom96-well plates. Twenty microliters of decreasing concentrations ofprednisolone were added and the plates were incubated for ninety-sixhours in a humidified incubator containing 5% CO₂ at 37° C. For thefinal six hours, ten microliters of 5 milligram per milliliter MTT(3-4,5-dimethylthiazol-2,5-diphenyl tetrazolium bromide) was added toeach experimental well. Drug resistance assays in cell lines wereperformed using between 0.25 and 2.5 million cells per milliliter.Assays were developed and performed as for patient sample assays. LC50values were determined as previously described (Holleman et al., 2004).Glucocorticoid resistant ALL was defined as having an LC50 greater thanor equal to 64 μM, whereas glucocorticoid sensitive cases were definedas having an LC50 less than 0.1 μM. Patients classified as sensitive toprednisolone (LC50<0.1 μM) had a significantly better treatment outcome,with a 5 year event free survival of 95.6% compared to 82.7% for otherpatients tested for prednisolone sensitivity on the Total XV protocol(p=0.01 log rank test).

CASP1 Enforced Expression

Full-length CASP1 (Origene, Rockville, Md., catalog #RC218364; GenBankAccession No. NM_033292) was subcloned into a lentiviral backbone(System Biosciences, Mountain View, Calif., catalog number CD527A-1), inframe with a T2A linked puromycin resistance gene. Lentivirus wasproduced in 293T cells and the Nalm6 leukemia cell line was transduced.72 hours post transduction, cells were selected with 2.5 micrograms permilliliter puromycin. Where indicated cells were treated with 10 μg/mLLPS (InvivoGen, San Diego, Calif.) followed 2 hours later by 5 mM ATP(Roche, Pleasanton, Calif.).

Expression of Mutant GR Without CASP1 Cleavage Sites

Doubly mutated GR (LLID (SEQ ID NO: 20) and IKQE (SEQ ID NO: 22); SEQ IDNO: 17) was subcloned into the pLX304 (Yang et al., 2011) lentiviralbackbone (Addgene plasmid 25890; Sigma-Aldrich, St. Louis, Mo.).Lentivirus was produced in 293T cells and NALM-6 leukemia cell lineswith CASP1 overexpression and shRNA directed towards CASP1 (SEQ ID NO:14) or a non-targeting shRNA (SEQ ID NO: 13) were transduced.Seventy-two hours post transduction, cells were selected withblasticidin.

Expression of GFP and CrmA

Green fluorescent protein (Addgene plasmid 15301) (Boehm et al., 2007)or CrmA (Addgene plasmid 11832; SEQ ID NO: 15) (Muzio et al., 1997) wassubcloned into the pLX304 (Yang et al., 2011) lentiviral backbone(Addgene plasmid 25890; Sigma-Aldrich, St. Louis, Mo.). Lentivirus wasproduced in 293T cells and NALM-6 leukemia cell lines with CASP1overexpression and shRNA directed towards CASP1 (SEQ ID NO: 14) or anon-targeting shRNA (SEQ ID NO: 13) were transduced. Seventy-two hourspost transduction, cells were selected with blasticidin.

Western Blotting

Cells were pelleted by centrifugation, washed once with PBS and lysedwith RIPA buffer or caspase-1 assay buffer (Enzo Life Sciences,Farmingdale, N.Y. catalog number KI-111), equal amounts of proteins(1-20 μg) separated by 4-12% Novex Bis-Tris gels (Life Technologies,Grand Island, N.Y.), and then transblotted to PVDF membranes (LifeTechnologies, Grand Island, N.Y.). Anti-GR (1:1000-1:10,000, BD catalognumber 611227), anti-DDK tubulin (1:1000, Origene catalog numberTA50011), anti-Bim (1:1000, Cell Signalling catalog number 2819) oranti-Tubulin (1:1000, Santa Cruz, sc-8035) were used as primaryantibodies followed by appropriate secondary HRP-conjugated IgG (1:1000,Dako) and immunocomplexes were visualized by chemiluminescence.

Glucocorticoid Induced Changes in Gene Expression

Nalm6 cells transduced with either empty vector (control) or CASP1containing lentiviral particles, were treated with or without 0.3 mMprednisolone for 24 hours. RNA was extracted and hybridized toAffymetrix PrimeView oligonucleotide microarrays and RMA processed.

To be included as a gene that was transactivated by glucocorticoids, thelevel of mRNA expression had to increase in control cells by at least1.5-fold in all four replicate experiments, by at least 2-fold in threeof four replicates and by at least 3-fold in one experiment. Similarly,to be included as a gene that was transrepressed by glucocorticoids, thelevel of mRNA expression had to decrease by at least 33% in all fourexperiments, by at least 50% in three of four experiments and by atleast 66% in one or more of the four replicate experiments.

Expression of CASP1 and NLRP3 in Leukemia Cells at the Time of DiseaseRelapse Versus at Diagnosis

Gene expression was assessed at diagnosis and relapse as previouslydescribed (29). This dataset (GEO accession: GSE28460) was MASS (22)processed using the affy (23) Bioconductor (24) R-project package andthen quantile normalized (30). A paired t-test was used for comparisonof the matching diagnosis and relapse data.

Results Higher Expression of CASP1 and NLRP3 in Glucocorticoid ResistantLeukemia

The de novo sensitivity of primary leukemia cells to prednisolone variedwidely among patients in each of the three independent cohorts of newlydiagnosed patients with ALL (FIGS. 1A-C). The present inventors foundthat CASP1 and NLRP3, both members of the NLRP3 inflammasome pathway,were expressed at a significantly higher level in glucocorticoidresistant primary ALL cells isolated from all three cohorts of patients(FIGS. 1D-F), with mean expression in resistant leukemia 1.6-fold higherthan in sensitive leukemia cells (Stouffer's meta-analysis of exactWilcoxon Mann-Whitney rank sum test p=9.5×10⁻⁷). Similarly, theexpression of NLRP3 was significantly higher (by 2.4-fold) inprednisolone-resistant leukemia cells in all three independent cohortsof patients (Stouffer's meta-analysis of exact Wilcoxon Mann-Whitneyrank sum test p=7.1×10⁻⁷; FIGS. 1G-I).

Lower Methylation of the CASP1 and NLRP3 Promoters is Associated withHigher CASP1 and NLRP3 Expression in Leukemia Cells

To determine whether epigenetic mechanisms influence CASP1 and NLRP3expression in leukemia cells, the relationship between CASP1 and NLRP3mRNA expression and methylation of their promoter regions in leukemiacells was assessed. This revealed a highly significant relationshipbetween the level of methylation of the CASP1 promoter and CASP1 mRNAexpression in ALL cells (Stouffer's meta-analysis of exact WilcoxonMann-Whitney rank sum test p=1.2×10⁻¹⁷; FIGS. 2A-C). In a subset ofpatients where matching germline DNA methylation was analyzed (n=55),CASP1 promoter methylation did not differ significantly (Paired t-testp=0.495) in germline DNA and leukemia cell DNA, although 10 patients hadsignificantly lower CASP1 promoter methylation in their ALL cells thantheir normal leukocytes, and the majority of these cases wereglucocorticoid resistant (n=7, 70%) suggesting CASP1 demethylation inglucocorticoid resistant ALL cells. Methylation of the promoter regionof NLRP3 was significantly greater in leukemia cell DNA than in germlineDNA (Paired t-test p=8.8×10⁻¹¹), and heterogeneity in leukemia cellmethylation was significantly related to NLRP3 leukemia cell expression(Stouffer's meta-analysis of exact Wilcoxon Mann-Whitney rank sum testp=5.5×10⁻⁵; FIGS. 2D-F). Categorization of patients using k-meansclustering of NLRP3 methylation and CASP1 methylation levelssignificantly distinguished prednisolone-sensitive (green symbols) fromprednisolone-resistant (red symbols) leukemia cells (Fisher's Exact testfor count data p=2.3×10⁻⁶; FIG. 2G). These results suggest thatdemethylation of CASP1 and NLRP3 in ALL cells leads to higher expressionof these genes in glucocorticoid resistant ALL cells.

CASP1 Cleaves the Glucocorticoid Receptor

Based on a prior report of CASP1 cleavage of the androgen receptor(Wellington et al., 1998), the structural similarity between theandrogen receptor and the glucocorticoid receptor, and the presentidentification of a putative CASP1 cleavage site (LLID) (SEQ ID NO: 20)in the transactivation domain of NR3C1 (FIG. 3A, upper panel), enzymaticassays were performed that revealed glucocorticoid receptor (NR3C1)cleavage by recombinant CASP1 (FIG. 3A, lower panel). Site-directedmutagenesis of the LLID motif (SEQ ID NO: 20) ablated cleavage of theglucocorticoid receptor at this location (FIG. 3B) and revealed asecondary CASP1 cleavage site in the GR (FIG. 3A, top). CASP1-inducedcleavage of the glucocorticoid receptor was inhibited by bothtetrapeptide inhibitors (FIG. 3A, lower panel) and small moleculeinhibitors (VX765 and VRT043198) of CASP1 (Boxer et al., 2010) (FIG.3C).

Full-length CASP1 was expressed in a human leukemia cell line (Nalm6) todetermine whether CASP1 over-expression increases resistance toglucocorticoids. Under standard conditions for activation of the NLRP3inflammasome and CASP1 (Ogura et al., 2006), cells transduced with emptyvector control virus showed no difference in their sensitivity toprednisolone, whereas cells transduced with CASP1-containing virusshowed markedly higher LC50 for prednisolone and dexamethasone (FIG.3E). On average, CASP1 over-expression increased resistance toprednisolone by 16.9-fold (Student's t-test p=5.7×10⁻⁶) anddexamethasone by 5.3-fold (Student's t-test p=1.2×10⁻⁹), with a 35%reduction in GR levels by 24 hours.

CASP1 Blunts Glucocorticoid-Induced Transcriptional Response

Because glucocorticoids regulate expression of target genes by bindingto the glucocorticoid receptor, triggering translocation into thenucleus and association with a glucocorticoid response element toincrease or decrease gene transcription, the present inventors assessedthe ability of glucocorticoids to influence gene expression in thepresence and absence of CASP1 enforced over-expression (FIGS. 4A, 4B).This revealed markedly diminished glucocorticoid-induced changes in geneexpression in cells with CASP1 enforced over-expression compared tocontrols. Additionally, induction of Bim protein levels, a knownglucocorticoid response gene was markedly blunted in CASP1overexpressing cells (FIG. 4A inset). Genome-wide assessment of changesin gene expression after glucocorticoid treatment, revealed 104 genesthat were induced following glucocorticoid treatment of control cells,64 (62%) of which had lower (by at least 25%) induction in CASP1over-expressing cells. Among these genes, the mean induction in controlcells was 7.8-fold, compared to only 4.4-fold in CASP1 over-expressingcells (Paired t-test p=0.015). Analysis of the sequences of these 104genes (including 5 kilobases up and downstream of each), revealed asignificant enrichment in these genes (54/104, 52%) of knownglucocorticoid response elements (GR binding motif, Table 1, motifs 1-9,(Dunham et al., 2012; Meijsing et al., 2009; Reddy et al., 2009)) or agenomic region shown by CHiP-seq to bind the GR (Reddy et al., 2009), ascompared to only 28% (1409/5000) of the randomly selected unchangedgenes (Fisher's Exact Test p=4.9×10⁻⁷). Inclusion of an additional motifcontaining the top two most frequent bases at each position of apreviously reported motif (Dunham et al., 2012) (motif 10, Table 1)increased the percentage of up-regulated genes with a glucocorticoidresponse element to 64% (67/104) among the transactivated genes.

Of the 28 genes whose expression was down-regulated followingglucocorticoid treatment of control cells, 23 (82%) of these genes hadlower (by at least 25%) down-regulation in CASP1 over-expressing cells.Among these genes, the mean reduction in control cells was 3.1-fold,compared to only 1.7-fold in CASP1 over-expressing cells (Paired t-testp=7.5×10⁻¹¹). Analysis of the sequence of these 28 genes, including 5kilobases up and downstream, revealed a significant enrichment (9/28,32%) of NF-κB binding motifs (a transcription factor known to interactwith an activated glucocorticoid receptor, Table 1, motif 11) or agenomic region shown by CHiP-seq to bind to the GR (Reddy et al., 2009),as compared to randomly selected unchanged genes (849/5000, 17%;Fisher's Exact Test p=0.04). The top 25 up-regulated and down-regulatedgenes in control cells and their changes in cells with CASP1 enforcedover-expression are depicted in FIG. 4, with the complete list ofglucocorticoid-modulated genes provided in the supplement (Tables 2-4).

CASP1 and NLRP3 Expression are Higher in Leukemia Cells at Relapse

To determine whether CASP1 and NLRP3 expression differed in leukemiacells at the time of disease recurrence, we examined the expressionlevels of CASP1 and NLRP3 in paired ALL cells obtained at diagnosis andrelapse from 49 patients (FIG. 8). This revealed significantly higherexpression of both CASP1 and NLRP3 in ALL cells at the time of relapsewhen compared to the corresponding samples obtained from the samepatients at diagnosis (Paired t-test p=3.2×10⁻⁴ and 4.2×10⁻³respectively).

CASP1 Inhibition Reverses CASP1-Induced GR-Cleavage and GlucocorticoidResistance

To determine whether inhibition of CASP1 could diminish CASP1-inducedglucocorticoid receptor cleavage and increase sensitivity toglucocorticoid treatment, shRNA (SEQ ID NO: 14) was used to knockdownoverexpressed CASP1. This revealed that knockdown of CASP1 expression by˜50% (FIG. 9A, shCASP1) reduced CASP1-induced GR cleavage (FIG. 9A,shCASP1), and markedly enhanced glucocorticoid sensitivity (12.8±4.7 μMvs. 570±423.2 μM, mean±S.E.M., 44-fold reduction in LC50, t-testp=0.028) (FIG. 9B, shCASP1) in CASP1 overexpressing ALL cells whencompared to cells transduced with scrambled non-targeting shRNA hairpin(FIG. 9A, shNT; SEQ ID NO: 13).

Likewise, when CASP1 overexpressing NALM-6 cells were transduced withthe gene encoding CrmA (cytokine response modifier A; SEQ ID NO: 15), aknown inhibitor of CASP1 catalytic activity (Garcia-Calvo et al., 1998;Komiyama et al., 1994; Ray et al., 1992), this blocked CASP1 induced GRcleavage (FIG. 9C) and markedly increased sensitivity to glucocorticoidtreatment (3.2±0.2 μM vs. 137.3±24.3 μM, mean±S.E.M., 43-fold reductionin LC50, t-test p=0.011, FIG. 9D) when compared to GFP transfectedcontrols.

Expression of a Glucocorticoid Receptor Mutated to Eliminate CASPCleavage Sites Markedly Attenuated CASP1 Induced Resistance toGlucocorticoids

Full-length wild-type GR (SEQ ID NO: 16) or a GR that had been mutatedto eliminate the CASP1 cleavage sites (i.e., alanines substituted forthe LLID (SEQ ID NO: 20) and IKQE (SEQ ID NO: 22) motifs identified inin vitro enzymatic assays; SEQ ID NO: 17), were overexpressed inleukemia cells overexpressing CASP1 in combination with eithershRNA-based knockdown of overexpressed CASP1 or non-targeting shRNAcontrol. Enforced expression of wild-type GR was unable to reverseCASP1-induced glucocorticoid resistance, whereas expression of a GRwithout the CASP1 cleavage sites markedly attenuated CASP1-inducedglucocorticoid resistance (9.4-fold reduction in LC50), restoringsensitivity to levels similar to cells with shRNA-based knockdown ofoverexpressed CASP1 (FIG. 10).

Discussion

The experiments disclosed herein have revealed a novel mechanism bywhich CASP1 and its activator NLRP3 modulate the biological andpharmacological effects of glucocorticoids via cleavage of theglucocorticoid receptor (GR). Glucocorticoids mediate their effects bybinding to the GR, causing it to translocate into the nucleus where itmodulates the expression of genes that contain a glucocorticoid responseelement (GRE). Low cellular levels of functional GR, due either to siRNAknockdown (Reddy et al., 2009; Wang et al., 2004), rare mutations in thehuman NR3C1 gene encoding the GR (Charmandari et al., 2008) orheterogeneity in the cellular levels of GR in leukemia cells byundefined mechanisms (Pui et al., 1984), have all been shown to alterresponse to glucocorticoids (i.e., lower functional GR associated withless response). However, prior to the current work, it was not knownthat CASP1 can cleave the GR and thereby reduce functional receptorlevels and modulate cellular response to glucocorticoids. The presentinventors have identified this new mechanism after first observing thatprimary leukemia cells that express high levels of CASP1 and NLRP3 arerelatively resistant to glucocorticoids. The present example also showshypomethylation of the CASP1 and NLRP3 genes in leukemia cellsexpressing higher levels of their messenger RNAs. As furtherdemonstrated herein, recombinant CASP1 cleaves the GR in itstransactivation domain, and forced over-expression of CASP1 coupled withits activation via the NLRP3 inflammasome causes human leukemia cells tobecome more resistant to glucocorticoids.The present example also showsthat over-expression of CASP1 significantly alters the transcriptionalresponse of human lymphoblasts to glucocorticoid treatment, causinglower induction of gene expression for 64 of 104 genes that were inducedin control cells after steroid treatment. Likewise, demonstrated hereinis markedly lower repression of gene expression for 23 of 28 genes thatwere down-regulated after glucocorticoid treatment of control cells. Thefact that not all GR-responsive genes were affected by CASP1over-expression likely reflects differences among genes in theirsensitivity to activated GR as a transcription factor and because therewas only a partial reduction in GR levels in CASP1 over-expressingcells. Whether glucocorticoids cause transactivation or transrepressionof a given gene is determined in part by the presence or absence ofeither a positive glucocorticoid response element, leading totransactivation or a negative GRE leading to transrepression, althoughthese GREs have not been fully elucidated. Glucocorticoids may alsorepress gene expression via binding of the activated GR to transcriptionfactors such as NF-κB and AP1 (McKay and Cidlowski, 1998; Teurich andAngel, 1995). Although the present inventors found a significantenrichment of positive GREs among transactivated genes and negative GREsin transrepressed gene, failure to find GREs in all genes likelyreflects their current state of incomplete definition. The broadspectrum of genes affected in CASP1 over-expressing cells indicates thatthese findings likely have broader implications, beyond the modulationof the antileukemic effects of glucocorticoids.

It is demonstrated herein that activation of the NLRP3 inflammasome islikewise required to activate over-expressed CASP1 in human leukemiacells, but that in a role distinct from its promotion of inflammation,activated CASP1 modulates the level of GR and cellular response toglucocorticoids. CASP1 is known to have pro-inflammatory effects,including the activation of inflammatory cytokines (e.g., interleukin 1βand interleukin 18). The findings disclosed herein raise the possibilitythat during inflammatory processes, CASP1 negatively regulatesanti-inflammatory GC signaling to further amplify its pro-inflammatoryeffects. Taken together, these results reveal a novel mechanism wherebyleukemia cells develop resistance to glucocorticoids via epigeneticchanges that cause over-expression of CASP1 and NLRP3, leading toenhanced CASP1-mediated cleavage of the GR, and diminished cellularresponse to glucocorticoids.

As further shown herein, overexpression of a glucocorticoid receptorthat was mutated to eliminate the CASP1 cleavage sites mitigated theeffects of CASP1 over-expression on leukemia cell sensitivity toglucocorticoids. Additionally, when CASP1 expression was knocked down inCASP1 over-expressing cells using shRNA, the present inventors were ableto reverse CASP1-induced GR cleavage and markedly increase sensitivityto glucocorticoids, suggesting CASP1 inhibition as a novel therapeuticstrategy. The present experiments showing that CrmA inhibition of CASP1catalytic activity restores sensitivity to glucocorticoids in CASP1over-expressing leukemia cells provide a further proof of principle forthis strategy.

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LIST OF SEQUENCES: SEQ ID SEQUENCE organism 1MADKVLKEKRKLFIRSMGEGTINGLLDELLQTRVLNKEEMEKVKRENATVMDK HomoTRALIDSVIPKGAQACQICITYICEEDSYLAGTLGLSADQTSGNYLNMQDSQGVLS sapiensSFPAPQAVQDNPAMPTSSGSEGNVKLCSLEEAQRIWKQKSAEIYPIMDKSSRTRLALIICNEEFDSIPRRTGAEVDITGMTMLLQNLGYSVDVKKNLTASDMTTELEAFAHRPEHKTSDSTFLVFMSHGIREGICGKKHSEQVPDILQLNAIFNMLNTKNCPSLKDKPKVIIIQACRGDSPGVVWFKDSVGVSGNLSLPTTEEFEDDAIKKAHIEKDFIAFCSSTPDNVSWRHPTMGSVFIGRLIEHMQEYACSCDVEEIFRKVRFSFEQPDGRAQMPTIERVTLTRCFYLFPGH 2MKMASTRCKLARYLEDLEDVDLKKFKMHLEDYPPQKGCIPLPRGQTEKADHVD HomoLATLMIDFNGEEKAWAMAVWIFAAINRRDLYEKAKRDEPKWGSDNARVSNPTV sapiensICQEDSIEEEWMGLLEYLSRISICKMKKDYRKKYRKYVRSRFQCIEDRNARLGESVSLNKRYTRLRLIKEHRSQQEREQELLAIGKTKTCESPVSPIKMELLFDPDDEHSEPVHTVVFQGAAGIGKTILARKMMLDWASGTLYQDRFDYLFYIHCREVSLVTQRSLGDLIMSCCPDPNPPIHKIVRKPSRILFLMDGFDELQGAFDEHIGPLCTDWQKAERGDILLSSLIRKKLLPEASLLITTRPVALEKLQHLLDHPRHVEILGFSEAKRKEYFFKYFSDEAQARAAFSLIQENEVLFTMCFIPLVCWIVCTGLKQQMESGKSLAQTSKTTTAVYVFFLSSLLQPRGGSQEHGLCAHLWGLCSLAADGIWNQKILFEESDLRNHGLQKADVSAFLRMNLFQKEVDCEKFYSFIHMTFQEFFAAMYYLLEEEKEGRTNVPGSRLKLPSRDVTVLLENYGKFEKGYLIFVVRFLFGLVNQERTSYLEKKLSCKISQQIRLELLKWIEVKAKAKKLQIQPSQLELFYCLYEMQEEDFVQRAMDYFPKIEINLSTRMDHMVSSFCIENCHRVESLSLGFLHNMPKEEEEEEKEGRHLDMVQCVLPSSSHAACSHGLVNSHLTSSFCRGLFSVLSTSQSLTELDLSDNSLGDPGMRVLCETLQHPGCNIRRLWLGRCGLSHECCFDISLVLSSNQKLVELDLSDNALGDFGIRLLCVGLKHLLCNLKKLWLVSCCLTSACCQDLASVLSTSHSLTRLYVGENALGDSGVAILCEKAKNPQCNLQKLGLVNSGLTSVCCSALSSVLSTNQNLTHLYLRGNTLGDKGIKLLCEGLLHPDCKLQVLELDNCNLTSHCCWDLSTLLTSSQSLRKLSLGNNDLGDLGVMMFCEVLKQQSCLLQNLGLSEMYFNYETKSALETLQEEKPELTVVFEPSW 3GTCGGGGAAGGTTTTGAGAAAGAAGGGTCCCTGGACAAGAACCTTGTCATTT HomoTCTGAGTGGCCGGTACCGAAAAGAGAGGAGGGAAGAACACACTGACTTTGAC sapiensTTTCATACGAAGCGGAAG 4CTCCTTTGACTTCAACTCCTTATCACTTCTCAAACAGGTTACAGTATCGGGGC HomoATTAGTTGCCCTGTTTTTAAAAGAACGACTACCCAGTTCTACCGTAGCACTTC sapiensACCAACAAGTGGCATT 5 CCTTGGAGATCAGACCTGTTGATAGCTGAAAACTGTTTGCTTTCsynthetic 6 GAAAGCAAACAGTTTTCAGCTATCAACAGGTCTGATCTCCAAGG synthetic 7CCTTGGAGATCAGACCTGTTGGCAGCTGAAAACTGTTTGCTTTC synthetic 8GAAAGCAAACAGTTTTCAGCTGCCAACAGGTCTGATCTCCAAGG synthetic 9CCTTGGAGATCAGACCTGGCGGCAGCTGAAAACTGTTTGCTTTC synthetic 10GAAAGCAAACAGTTTTCAGCTGCCGCCAGGTCTGATCTCCAAGG synthetic 11CCTTGGAGATCAGACGCGGCGGCAGCTGAAAACTGTTTGCTTTC synthetic 12GAAAGCAAACAGTTTTCAGCTGCCGCCGCGTCTGATCTCCAAGG synthetic 13CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGT synthetic TTTT 14CCGGCTACAACTCAATGCAATCTTTCTCGAGAAAGATTGCATTGAGTTGTAGT synthetic TTTT 15ATGGATATCTTCAGGGAAATCGCATCTTCTATGAAAGGAGAGAATGTATTCAT OrthopoxTTCTCCACCGTCAATCTCG virusTCAGTATTGACAATACTGTATTATGGAGCTAATGGATCCACTGCTGAACAGCT vacciniaATCAAAATATGTAGAAAAGGAGGCGGACAAGAATAAGGATGATATCTCATTCAAGTCCATGAATAAAGTATATGGGCGATATTCTGCAGTGTTTAAAGATTCCTTTTTGAGAAAAATTGGAGATAATTTCCAAACTGTTGACTTCACTGATTGTCGCACTGTAGATGCGATCAACAAGTGTGTTGATATCTTCACTGAGGGGAAAATTAATCCACTATTGGATGAACCATTGTCTCCAGATACCTGTCTCCTAGCAATTAGTGCCGTATACTTTAAAGCAAAATGGTTGATGCCATTTGAAAAGGAATTTACCAGTGATTATCCCTTTTACGTATCTCCAACGGAAATGGTAGATGTAAGTATGATGTCTATGTACGGCGAGGCATTTAATCACGCATCTGTAAAAGAATCATTCGGCAACTTTTCAATCATAGAACTGCCATATGTTGGAGATACTAGTATGGTGGTAATTCTTCCAGACAATATTGATGGACTAGAATCCATAGAACAAAATCTAACAGATACAAATTTTAAGAAATGGTGTGACTCTATGGATGCTATGTTTATCGATGTGCACATTCCCAAGTTTAAGGTAACAGGCTCGTATAATCTGGTGGATGCGCTAGTAAAGTTGGGACTGACAGAGGTGTTCGGTTCAACTGGAGATTATAGCAATATGTGTAATTCAGATGTGAGTGTCGACGCTATGATCCACAAAACGTATATAGATGTCAATGAAGAGTATACAGAAGCAGCTGCAGCAACTTGTGCGCTGGTGGCAGACTGTGCATCAACAGTTACAAATGAGTTCTGTGCAGATCATCCGTTCATCTATGTGATTAGGCATGTCGATGGCAAAATTCTTTTCGTTGGTAGATATTGCTCTCCAACAACTAATTAA 16MDSKESLTPGREENPSSVLAQERGDVMDFYKTLRGGATVKVSASSPSLAVASQS HomoDSKQRRLLVDFPKGSVSNAQQPDLSKAVSLSMGLYMGETETKVMGNDLGFPQQ sapiensGQISLSSGETDLKLLEESIANLNRSTSVPENPKSSASTAVSAAPTEKEFPKTHSDVSSEQQHLKGQTGTNGGNVKLYTTDQSTFDILQDLEFSSGSPGKETNESPWRSDLLIDENCLLSPLAGEDDSFLLEGNSNEDCKPLILPDTKPKIKDNGDLVLSSPSNVTLPQVKTEKEDFIELCTPGVIKQEKLGTVYCQASFPGANIIGNKMSAISVHGVSTSGGQMYHYDMNTASLSQQQDQKPIFNVIPPIPVGSENWNRCQGSGDDNLTSLGTLNFPGRTVFSNGYSSPSMRPDVSSPPSSSSTATTGPPPKLCLVCSDEASGCHYGVLTCGSCKVFFKRAVEGQHNYLCAGRNDCIIDKIRRKNCPACRYRKCLQAGMNLEARKTKKKIKGIQQATTGVSQETSENPGNKTIVPATLPQLTPTLVSLLEVIEPEVLYAGYDSSVPDSTWRIMTTLNMLGGRQVIAAVKWAKAIPGFRNLHLDDQMTLLQYSWMFLMAFALGWRSYRQSSANLLCFAPDLIINEQRMTLPCMYDQCKHMLYVSSELHRLQVSYEEYLCMKTLLLLSSVPKDGLKSQELFDEIRMTYIKELGKAIVKREGNSSQNWQRFYQLTKLLDSMHEVVENLLNYCFQTFLDKTMSIEFPEMLAEIITNQIPKYSNGNI KKLLFHQK 17MDSKESLTPGREENPSSVLAQERGDVMDFYKTLRGGATVKVSASSPSLAVASQS SyntheticDSKQRRLLVDFPKGSVSNAQQPDLSKAVSLSMGLYMGETETKVMGNDLGFPQQGQISLSSGETDLKLLEESIANLNRSTSVPENPKSSASTAVSAAPIEKEFPKTHSDVSSEQQHLKGQTGTNGGNVKLYTTDQSTFDILQDLEFSSGSPGKETNESPWRSDAAAAENCLLSPLAGEDDSFLLEGNSNEDCKPLILPDTKPKIKDNGDLVLSSPSNVTLPQVKTEKEDFIELCTPGVAAAAKLGTVYCQASFPGANIIGNKMSAISVHGVSTSGGQMYHYDMNTASLSQQQDQKPIFNVIPPIPVGSENWNRCQGSGDDNLTSLGTLNFPGRTVFSNGYSSPSMRPDVSSPPSSSSTATTGPPPKLCLVCSDEASGCHYGVLTCGSCKVFFKRAVEGQHNYLCAGRNDCIIDKIRRKNCPACRYRKCLQAGMNLEARKTKKKIKGIQQATTGVSQETSENPGNKTIVPATLPQLTPTLVSLLEVIEPEVLYAGYDSSVPDSTWRIMTTLNMLGGRQVIAAVKWAKAIPGFRNLHLDDQMTLLQYSWMFLMAFALGWRSYRQSSANLLCFAPDLIINEQRMTLPCMYDQCKHMLYVSSELHRLQVSYEEYLCMKTLLLLSSVPKDGLKSQELFDEIRMTYIKELGKAIVKREGNSSQNWQRFYQLTKLLDSMHEVVENLLNYCFQTFLDKTMSIEFPEMLAEIITNQIPKYSNGN IKKLLFHQK

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

All patents, applications, publications, test methods, literature, andother materials cited herein are hereby incorporated by reference intheir entirety as if physically present in this specification.

1-79. (canceled)
 80. A method for treating glucocorticoid resistance orincreasing glucocorticoid sensitivity in a subject in need thereof,wherein the expression level of CASP1 gene and/or NLRP3 gene in cells ofthe subject is increased as compared to a correspondingglucocorticoid-sensitive control, which method comprises administeringto the subject a therapeutically effective amount of an inhibitor ofCASP1.
 81. The method of claim 80, wherein the method further comprises(i) determining the expression level of CASP1 gene and/or NLRP3 gene inthe cells from the subject or (ii) determining the methylation level ofCASP1 gene promoter and/or NLRP3 gene promoter in the cells from thesubject.
 82. The method of claim 80, wherein the expression level ofCASP1 gene and/or NLRP3 gene in said cells is increased by at least1.5-fold as compared to the corresponding expression level in theglucocorticoid-sensitive control.
 83. The method of claim 80, whereinthe inhibitor of CASP1 directly inhibits expression or function ofCASP1.
 84. The method of claim 80, wherein the inhibitor of CASP1directly inhibits expression or function of NLRP3.
 85. The method ofclaim 80, wherein the inhibitor of CASP1 inhibits NLRP3 inflammasomeformation or NLRP3 inflammasome activity.
 86. The method of claim 80,wherein the inhibitor of CASP1 is selected from the group consisting ofsiRNA, shRNA, z-VAD-DCB, Ac-YVAD-CHO, Ac-YVAD-chloromethylketone,cytokine response modifier A (crmA), Pralnacasan (VX-740), IDN-6556,VX-765, VRT-043198, ML132, and SNAP.
 87. The method of claim 80, whereinthe administration of the inhibitor of CASP1 results in an increasedglucocorticoid sensitivity.
 88. The method of claim 80, wherein thesubject is human.
 89. A method of treating a disease in a subject inneed thereof, wherein the disease is treated with glucocorticoids andcan acquire glucocorticoid resistance accompanied by an increasedexpression level of CASP1 gene and/or NLRP3 gene in cells of the subjectas compared to a corresponding glucocorticoid-sensitive control, whichmethod comprises administering to the subject (i) a therapeuticallyeffective amount of an inhibitor of CASP1 and (ii) a therapeuticallyeffective amount of a glucocorticoid.
 90. The method of claim 89,wherein the disease is an inflammatory or an autoimmune disease.
 91. Themethod of claim 89, wherein the disease is a glucocorticoid-resistantasthma.
 92. The method of claim 89, wherein the method further comprises(i) determining the expression level of CASP1 gene and/or NLRP3 gene inthe cells from the subject or (ii) determining the methylation level ofCASP1 gene promoter and/or NLRP3 gene promoter in the cells from thesubject.
 93. The method of claim 89, wherein the expression level ofCASP1 gene and/or NLRP3 gene in said cells is increased by at least1.5-fold as compared to the corresponding expression level in theglucocorticoid-sensitive control.
 94. The method of claim 89, whereinthe inhibitor of CASP1 directly inhibits expression or function ofCASP1.
 95. The method of claim 89, wherein the inhibitor of CASP1directly inhibits expression or function of NLRP3.
 96. The method ofclaim 89, wherein the inhibitor of CASP1 inhibits NLRP3 inflammasomeformation or NLRP3 inflammasome activity.
 97. The method of claim 89,wherein the inhibitor of CASP1 is selected from the group consisting ofsiRNA, shRNA, z-VAD-DCB, Ac-YVAD-CHO, Ac-YVAD-chloromethylketone,cytokine response modifier A (crmA), Pralnacasan (VX-740), IDN-6556,VX-765, VRT-043198, ML132, and SNAP.
 98. The method of claim 89, whereinthe administration of the inhibitor of CASP1 results in an increasedglucocorticoid sensitivity.
 99. The method of claim 89, wherein thesubject is human.